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lymphoma cell line L5178Y and marrow stromal cells
Leukemia Research 22 (1998) 805 – 815
CD45 partially mediates heterotypic adhesion between murine leukemia/lymphoma cell line L5178Y and marrow stromal cells Harinder S. Juneja a,*, Frank C. Schmalstieg b, Sang Lee a, Jian Chen a a
Department of Internal Medicine, Di6ision of Hematology/Oncology, Uni6ersity of Texas Health Science Center, Room 5.016 MSB, 6431 Fannin Street, Houston, TX 77030, USA b The Department of Pediatrics, Uni6ersity of Texas, Medical Branch, Gal6eston, Gal6eston, TX, USA Received 17 November 1997; accepted 6 March 1998
Abstract We raised mAbs to whole L5178Y leukemia/lymphoma (LL) cells to identify adhesion proteins involved in adherence between LL cells and marrow stromal cells. One mAb, 4C, and its subclones 4C.1 and 4C.2 inhibited adherence of L5178Y LL cells to MLT, a nontransformed murine marrow stromal cell line. These MoAbs are directed against CD45RA. Control anti-CD45 mAbs and isotype mAbs were non-inhibitory. Other anti-CD45 mAbs, M1/9.3, RA3-3A1/6.1 and RA3-2C2/1 do not compete with mAb 4C.1 for binding to the L5178Y cell surface, but mAb 4C.1 competes for binding of mAb RA3-2C2/1. Effects of mAb 4C on tyrosine-phosphatase activity of CD45 in L5178Y cells are minimal, suggesting direct involvement of CD45 as an adhesion protein. © 1998 Elsevier Science Ltd. All rights reserved. Keywords: CD45; Marrow stromal cells; Adhesion; Leukemia; Lymphoma; Metastasis; Cell adhesion; Lymphocytes; Integrins
1. Introduction Marrow stromal cells are essential for sustained hematopoiesis both in vivo and in vitro [1 – 6]. In addition, factor-dependent hematopoietic cell lines can become factor independent when co-cultured in vitro with marrow stromal cells for several weeks to months [7 – 11]. During normal hematopoiesis in long-term cultures and the development of leukemic changes in the factor-dependent cell lines, the hematopoietic cells are in close apposition or adherent to the marrow stromal cells [9,12–14]. Physical apposition of pre-B-cells and marrow stromal cells is also essential for long-term B-cell lymphopoiesis in vitro [3,15]. We have previously utilized two murine leukemia/lymphoma (LL) cell lines, L5178Y and L1210, and a nontransformed murine marrow stromal cell line (MLT) to dissect the mechanism of LL cell adherence to marrow stromal cells [16]. MLT is a unique cell line that lacks expression of the
* Corresponding author. 0145-2126/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved. PII: S0145-2126(98)00070-8
intercellular adhesion molecule, ICAM-1. Studies with inhibitory monoclonal antibodies (mAbs) indicated that CD44, CD11a and b, CD18 and CD62 are not involved in LL cell adherence to MLT. Neoglycoprotein probes, mannosyl-bovine serum albumin (BSA) and galactosylBSA caused a partial, but significant, inhibition of LL cell adherence to MLT. In contrast, fucosyl-BSA had no inhibitory effects in this system. These data suggested that: (i) adherence of LL cells to marrow stromal cells involves a lectin mechanism with mannosyl and galactosyl specificities; and (ii) other mechanisms of adherence, not yet described, were also important in this system. To determine the role of as yet undescribed adhesion proteins, we raised rat mAbs to whole L5178Y LL cells. We present data on characterization of one mAb, 4C and its subclones, 4C.1 and 4C.2, which inhibited adherence of L5178Y LL cells to MLT. mAbs PS/2 (anti-CD49d) and MK/2 (anti-CD106) were also utilized to determine the role of integrin VLA-4 and its ligand vascular cell adhesion molecule-1 (VCAM-1), in this system.
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2. Materials and methods
2.1. Marrow stromal cell line A previously described murine marrow stromal cell line (MLT) was utilized. These marrow stromal cells are fibroblast-like in appearance, peroxidase and leukocyte alkaline phosphatase negative and non-specific esterase positive. Importantly, the MLT cell line does not express intercellular adhesion molecule-1 [16]. It was derived from long-term cultures of marrow obtained from DBA/2J mice and is passaged in vitro bi-weekly in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% fetal calf serum. The MLT cells were grown to confluence in 24-well plates for the adherence experiments.
2.2. Leukemia/lymphoma cell lines L5178Y, a lymphoma cell line originally developed from methylcholanthrene-treated DBA strain mice, was obtained from the NCI (DCT tumor repository, Frederick, MD). In our hands, the L5178Y cell line obtained from the repository stains positive with mAb 14.8 (TIB 164-ATCC, Rockville, MD) [17] which reacts with an epitope on B220 glycoprotein expressed by murine B-cells, B-cell precursors and a subpopulation of peripheral T-cells. The L5178Y cells are passaged intraperitoneally in DBA/2J mice (Harlan Sprague– Dawley, Houston, TX). At monthly intervals, fresh in vitro cultures of L5178Y cells are established from in vivo passaged cells in an attempt to maintain a constant phenotype. Several pre-B-, B-, T-cell and monocyte macrophage cell lines, listed in Table 1, were used in Table 1 Distribution of 4C epitope on myeloid and lymphoid cell lines Cell lines
Type
4C.1
M1/9.3HL
P388 L1210 70Z A20 L5178Y S49.1 S49 TIM.4 CTLL-2 P1793 Wehi 274-1 Wehi 3 IC-21
Lymphoblast Early lymphoblast Pre-B B-cell B-cell T Thy 1.2 T Thy-1− T TL(−) T T Monocyte Mono-Macro Macrophage
70/4 1000/11 300/10 16/1 300/15 8/6 9/6 13/5 20/20 6/3 30/3 3/3 5/3
ND 400/5 ND ND 1500/15 200/6 700/6 130/5 ND 600/3 120/3 130/3 150/3
Several cell lines were examined for expression of the M1/9.3HL and 4C epitopes using indirect immunofluorescence techniques. The stained cells were analyzed on a FACScan. The table shows the mean fluorescence channel under test/control conditions. Controls represent staining with only the FITC-labelled goat anti-rat antibody. All cell lines were obtained from ATCC (Rockville, MD). ND, not done.
characterization of the MoAb 4C. A set of well characterized parental CD45 + , mutant CD45 − , and revertant CD45 + T-cell lymphoma lines (BW5147G.1.4OUAR.1, BW5147 (T200 − a) 5.1 and BW5147 (Rev)1.1, respectively) were kindly provided by Dr Robert Hyman (Salk Institute, La Jolla, CA) [18]. These lymphoma cell lines were also grown in IMDM supplemented with 10% FCS.
2.3. Production of mAb against L5178Y cells Intact L5178Y cells were injected i.p. into 12–16 week old Wistar–Furth rats (Jackson Laboratories, Bar Harbor, ME). Hybridomas were generated by fusing nonsecretory myeloma cells (P3× 63Ag8.653) (ATCC, Rockville, MD) with spleen cells from the immunized rats, using standard protocols [19,20]. The hybridomas were first screened for reactivity against cell surface antigens on L5178Y cells by a cell-ELISA technique [20,21]. Conditioned medium, from clones positive on the ELISA screen, was used in an adherence assay to select hybridomas that inhibited adherence of L5178Y cells to MLT monolayers. A single mAb 4C and its subclones, 4C.1 and 4C.2, were selected for characterization (see below).
2.4. Heterotypic adherence between L5178Y and T-lymphoma cell lines and MLT Adherence of 51chromium (Cr) labelled L5178Y cells or T lymphoma cell lines to MLT monolayers was determined using previously described methodology [22]. Briefly, varying numbers of radiolabelled L5178Y cells or T lymphoma cells were added to MLT monolayers grown in IMDM and 10% FCS. After a 2 h incubation at 37°C in the presence of 75% N2, 20% O2 and 5% CO2, the wells were washed gently with PBS (pH 7.2) three times, and the adherent cells were lysed with 1N sodium hydroxide. The radioactivity recovered from the adherent layer was used to calculate the LL cell adherence to MLT. The quantitative adherence of L5178Y LL cells to MLT cells has not changed over a period of 4 years.
2.5. Effect of mAbs on heterotypic adherence of L5178Y cells to MLT L5178Y cell adherence to MLT was determined as described above in the presence or absence of a test mAb. mAbs used were 4C.1, M1/9.3HL (anti-T200) and anti-CD45RA mAb’s RA3-2C2/1 and RA3-3A1/ 6.1 [20,23–25]. mAb M1/9.3HL, directed against an epitope expressed by all lines of leukocyte differentiation, was either obtained from Boehringer Mannheim (Indianapolis, IN) or prepared from a hybridoma (TIB122) obtained from ATCC. F(ab)2 fragment of
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mAb 4C.1 was prepared by previously described methods [26]. Monoclonal isotype immunoglobulins (Chemicon, Temecula, CA) were used as isotype controls. Sodium bi-sulphate precipitated and dialyzed Ig form of mAbs PS/2 and MK/2 (ATCC Rockville, MD); [27,28] were utilized at a concentration of 10 mg ml − 1 in similar experiments to determine the role of integrin CD49d and its ligand, CD106, in our system. The adherence of L5178Y cells to MLT under the test conditions is represented as percent of controls. Percent adherence is presented as the mean (9 1 S.D.). All experiments were done in triplicate and repeated 3–21 times. Effect of mAb 4C.1 on heterotypic adherence L5178Y cells and T-cell lymphoma cell lines (BW5147G.1.4OUAR.1, BW5147 (T200 − a) and BW5147 (Rev)) to MLT was also determined in concurrent experiments.
2.6. Cell surface iodination of L5178Y cells L5178Y cells (2× 107) were suspended in 1 ml of PBS, pH 7.4 and placed on ice. All subsequent steps were performed on ice. A total of 5 U of lactoperoxidase and 5 mCi of 125I were then added followed by 20 ml of 0.06% H2O2. After 5 min, 10 ml of 0.06% H2O2 was added and the incubation was carried out for an additional 5 min. The cells were washed three times and subsequently lysed in 150 ml of buffer containing 0.5% NP-40, 5 mM PO4, 150 mM NaCl, 10 mM pyrophosphate, 25 mM NaF, 1 mM ZnC12, 5 mM EDTA, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 10 mg ml − 1 each of soybean trypsin inhibitor, anti-papain, leupeptin, and pepstatin A, and 20 mM HEPES, pH 7.4 [29]. The cell lysate was centrifuged for 1 min to remove DNA. The supernatant was removed and saved.
2.7. Immunoprecipitation of L5178Y cell membrane lysate by mAbs, 4C.2 and M1 /9.3HL Affinity purified mAbs 4C.2 and M1/9.3HL were prepared by using a commercial kit (ImmunoPure Ag/ Ab Immobilization Kit, Pierce, Rockford, IL). The lysed membrane preparation was precleared as follows: Irrelevant 50 ml of nonimmune rabbit serum was added to each supernatant and the sample incubated with rocking overnight at 4°C. After the preclearing procedure, the radio-labelled cell lysate was aliquoted into two portions and 1 mg of 4C.2 or M1/9.3HL antibodies, previously bound to 0.2 ml suspension of protein G sepharose A beads (2.4 mg IgG binding capacity) (Sigma), was added to the aliquots. Incubation was carried out for 30 min at 4°C. These incubations were repeated four times and then the alternate antibody was added to each supernatant. The protein G sepharose
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beads containing the immune complexes were washed 5× with PBS. Sample buffer (15 ml, 2.3% SDS and 0.36 M 2-ME) was added to the beads, without boiling. The iodinated proteins thus recovered from the beads were subjected to electrophoresis on 10% polyacrylamide gels. After drying, the gels were placed with X-OMAT film (Eastman Kodak, Rochester, NY) with intensifying screens at − 70°C for 7.5 h.
2.8. Competiti6e binding studies In order to determine if other anti CD45 mAbs were directed against the same epitope on the CD45 molecule as 4C, competitive binding studies were performed in the following manner. Affinity purified mAbs were fluorescein labelled by standard techniques [30]. L5178Y cells were suspended in 200 ml of PBS+1% BSA and treated with 10 mg ml − 1 of unlabelled affinity purified mAbs M1/9.3HL, 4C.2, RA3-2C2/1 and RA3A1/6.1 for 1 h at room temperature followed by treatment with 1 mg of FITC labelled 4C.2 for 1 h. Cells were washed three times with 5 ml PBS+ 1% BSA and resuspended in 1 ml buffer and analyzed on a FACScan (Becten-Dickinson, Mountain View, CA) as described below. To determine further similarities or differences between the epitopes for mAbs 4C.2 and M1/9.3 HL, untreated L5178Y cells and L5178Y cells treated with 0.5% trypsin-EDTA (Sigma, St. Louis, MO) for 15 min were stained with FITC labelled 4C.2 or FITC labelled isotype Ig.
2.9. Expression of 4C.2 and M1 /9.3HL epitopes in hematopoietic tissues and on hematopoietic cell lines Peripheral blood and the hematopoietic tissues (bone marrow, spleen and thymus) were obtained from 8–10 week old DBA/2 mice and single cell suspensions prepared. Single cell suspensions from the tissues were prepared by cutting the tissues into small pieces, homogenization of the tissue in a syringe and rinsing out of cells released. The cell suspension was then allowed to stand for 5 min and the supernatant passed through a 27 gauge needle to remove large clumps and fibrous tissue. A panel of murine hematopoietic cell lines of myeloid, monocytic and lymphoid origin were obtained from ATCC (Table 1). A total of 106 cells from each of the tissues or the cell lines were suspended in PBS+1% BSA in a 2 ml microfuge tube and incubated with 4C.1, M1/9.3HL or an isotype mAb for 60 min followed by incubation with an FITC labelled goat anti-rat Ig antibody for 45 min. After washing the cells twice with 2 ml PBS+ 1% BSA, the cells were analyzed on a FACScan utilizing the LYSIS software (BD, CA). The analysis were performed in the Automated Cytometry core facility at the University of Texas M.D. Anderson Cancer Center as previously described [31].
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Fig. 1. mAbs 4C.1 and PS/2 inhibit L5178Y adherence to MLT. 51Cr-labelled L5178Y cells were incubated with each of the mAbs diluted in IMDM+ 2% BSA, separately, for 1 h and allowed to adhere to MLT monolayers in the presence of the same mAb for 2 h. At the end of the assay, the L5178Y cell viability, by dye-exclusion techniques, was ]95%. Also, no homotypic aggregation of L5178Y cells was noted under the inverted microscope. Adherence of L5178Y cells to MLT in presence of each mAb is presented as a percentage of the adherence of L5178Y cells to MLT under control conditions. * P B0.05 on a two-sample analysis by Student’s t-test (Statgraphics, Rockville, MD) on comparison with the controls. (A) Test mAbs included 4C.1 conditioned medium (undiluted) or ascites (1:100). F(ab)2 fragment of mAb4C.1 (10 mg ml − 1) and rat IgG 2a isotype control (MEL-14). Adhesion of L5178Y cells under control conditions (IMDM + 2% BSA) ranged from 13.6 95.5 to 22.5 9 12.4%. (B) Effect of other anti-CD45 mAbs, i.e. M1/9.3HL (1 ×conditioned medium), RA3-2C2/1 (Ig 10 mg ml − 1) and RA3A1/6.1 (Ig 10 mg ml − 1) and PS/2 (Ig 10 mg ml − 1) is represented as a percentage of isotype controls (rat IgM, rat IgG 2a and rat IgG 2b, respectively). Data with mAb MK/2 is represented as a percent of control with IMDM +2% BSA. Adhesion of L5178Y to MLT in the controls ranged from 18.9 9 6.8 to 24.2 9 2.4%. Isotype mAbs were used as Ig form (10 mg ml − 1). (n), number of observations.
2.10. Tyrosine phosphorylation in L5178Y cells L5178Y cells (1 × 107) were suspended in 500 ml of RPMI-1640 in each of 18 tubes. Anti-CD45 (4C.1) (14 mg) was added to certain tubes and incubated for 1 min at room temperature. PMA (0.5 mg) was added to all but the control samples, and the tubes brought to 37°C. At 0, 2, 5, 10, 20 and 30 min, 500 ml of ice-cold PBS containing 500 mM sodium orthovanadate was added and the cells washed once in this medium. Following the wash, the cells were pelleted and lysed in 150 ml of cold lysis buffer (1% NP-40, 5 mM PO4, 150 mM NaC1, 10 mM pyrophosphate, 1 mM sodium orthovanadate, 25 mM NaF, 1 mM ZnC12, 5 mM EDTA, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 10 mg ml − 1 each of soybean trypsin inhibitor, leupeptin, and pepstain A, and 20 mM HEPES, pH 7.4) for 20 min on ice. The lysates were centrifuged to remove nuclei and cell debris and immediately added to 2 ×SDS sample buffer (reducing) and boiled for 2 min. The samples were subjected to electrophoresis on 10% polyacrylamide gels, and after electrophoretic transfer to nitrocellulose, blocked in PBS containing 5% BSA, 0.05% azide, and 500 mM sodium orthovanadate for 4 h
at 4°C. The phosphotyrosine containing proteins were reacted with murine monoclonal anti-phosphotyrosine antibody (Sigma). The second antibody was rabbit anti-murine immunoglobulin conjugated to horseradish peroxidase. Detection was accomplished by addition of 0.025% H2O2 and 4-chloro-1-naphthol as previously described [32]. The images were digitalized and densitometric analyses performed.
3. Results Of the 250 clones that were positive on the ELISA screen, only one mAb, 4C, and its subclones 4C.1 and 4C.2, significantly inhibited adherence of L5178Y cells to MLT. Using an isotyping kit (Binding Site, San Diego, CA) mAb 4C and its subclones were identified as rat IgG2a proteins. Data from experiments with subclone 4C.1 is shown in Fig. 1. F(ab)2 fragments of 4C.1 also inhibited L5178Y adherence to MLT by 23.59 5.1% (Fig. 1). It is important to note that isotype-control (IgG2a) antibody, MEL-14, did not cause a decrease in L5178Y adherence to MLT. In contrast, anti-CD45 mAbs M1/9.3HL, RA3-2C2/1 and RA3-
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Fig. 2. Inhibitory activity of mAb 4C on heterotypic adherence between L5178Y and marrow stromal line (MLT) is specific. 51Cr labelled L5178Y and T lymphoma cell lines (BW5147G.1.4OUAR.1, BW5147 (T200 − a) and BW5147 (Rev)) were incubated in 4C.1CM (1 × ) for 1 h and then allowed to adhere to MLT monolayers for 2 h in the continuous presence of mAb 4C.1. At end of 2 h, the percent of adherence LL cells was determined as described in Section 2. Adherence is represented as the percent of the total number of cells (input) added to the MLT monolayer. The T-cell lymphoma cell lines show a greater adherence to MLT than L5178Y cells. Adherence of L5178Y to MLT is significantly inhibited by mAb 4C while the adherence of the T lymphoma cells to MLT was unaffected.
3A1/6-1 did not inhibit the adherence of L5178Y cells to MLT. mAb PS/2 (anti-CD49d) caused a significant reduction, while mAb MK/2 (anti-CD106) caused an increase in L5178Y adherence to MLT (Fig. 1). Notably, mAb 4C did not inhibit the adherence of the CD45 + , mutant and revertant T lymphoma cell lines to MLT, suggesting that its effect on L5178Y-MLT interaction is not nonspecific, but rather, a specific effect (Fig. 2).
3.1. Comparison of 4C and M1 /9.3HL Radio-iodination of L5178Y cells was carried out to compare the antigen specificities of mAbs 4C.2 and M1/9.3HL (Fig. 3). Sequential clearing of radio-labelled cell lysates were performed with each antibody and final immunoprecipitation attempted with the alternative antibody. Both antibodies recognized the same 230 – 240 kDa protein, establishing 4C as an anti-CD45 antibody.
3.2. Competiti6e binding to the CD45 on the cell surface by 4C and other known anti-CD45 mAbs Competition experiments demonstrated that mAbs M1/9.3HL, RA3-2C2/1 and RA3-3A1/6.1 do not compete for binding of FITC-labelled mAb 4C.2 to L5178Y cells. Unlabelled 4C.2, at 10× concentration, competes
with binding of FITC labelled 4C.2 (Fig. 4, top row). MoAb 4C.2 effectively competed with binding of FITC labelled RA3-2C2/1 (Fig. 4, middle row) but not with FITC labelled RA3-3A1/6.1 (Fig. 4 bottom row). MoAb RA3-2C2/1 is directed against an epitope dependent on the expression of the first alternatively spliced exon (exon A) of the CD45 molecule and expressed on B-cells and at a lower level on CD8 + T-cells, while MoAb RA3-3A1/6.1 is also directed against the RA isoform of CD45 but the exact epitope has not been identified [33]. MoAb M1/9.3HL did not compete with 4C.2, RA3-2C2/1 or RA3-A1/6.1. mAb 4C is directed against a trypsin-sensitive epitope on CD45, while M1/ 9.3HL is directed against a trypsin-resistant epitope on CD45 (Fig. 5). Expression of the M1/9.3HL and 4C epitopes on hematopoietic cell lines is shown in Table 1. The M1/ 9.3HL epitope was present on pre-B-, pro B-, B-, Tand monocyte-macrophage cell lines. In contrast, the 4C epitope was present on pre-B, B and non-B-non-T lymphoid cells but not on the majority of the macrophages-monocytes or T-cells. Only one T-cell line, TIMI-4, was very weakly positive on FACScan analysis and showed a patchy membrane staining pattern on indirect immunofluorescence microscopy. Peripheral blood lymphocytes stained strongly positive with mAb M1/9.3HL, while monocytes and neutrophils had two subgroups, one weak and the other strongly
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positive. In contrast, staining with mAb 4C.2 revealed two lymphocyte subpopulations, one negative and the other positive. A vast majority (#85%) of granulocytes and monocytes did not exhibit the 4C epitope. A small percentage of monocytes may be positive, which is in keeping with expression of 4C epitope on some of the monocyte-macrophage cell lines. This data is consistent with the distribution of the 220 kd isoform of CD45. A single peak of M1/9.3HL positive cells was observed on cells isolated from the bone marrow and spleen. Based on the 4C epitope expression, both the spleen and the bone marrow cells could be divided into a positive and a negative subpopulation. Lymphoid cells of the thymus were negative for 4C, but M1/9.3HL positive. Thus, 4C and M1/9.3HL epitopes are distributed differentially on hematopoietic cells (data not shown).
3.3. Influence of 4C.1 on tyrosine phosphorylation in L5178Y cells In order to determine whether 4C had an effect on tyrosine phosphorylation, and hence cell signaling in L5178Y cells, kinetics of PMA-induced phosphorylation with and without 4C.1 was carried out. PMA induced an immediate increase in phosphorylation of 130, 110, and 80 kDa proteins in L5187Y cells (Fig. 6). 4C.1 had little effect on the kinetics of phosphorylation of the 80 kDa protein (Fig. 7).
Fig. 3. mAb 4C.2 immunoprecipitates the same protein as mAb M1/9.3HL, i.e. CD45 from L5178Y cell membrane. Membrane proteins of L5178Y cells were iodinated with 125I. The membrane was lysed and precleared with nonimmune rabbit serum. Aliquots of the cell membane lysate were treated with protein G-Sepharose A bead bound mAbs 4C.2 or M1/9.3HL. These incubations were repeated × 4 and then alternate antibody was added to each supernatant. The protein G-sepharose beads containing the immune complexes were washed repeatedly with PBS. The immune complexes were removed by treatment with SDS/2-ME buffer, without boiling. The iodinated proteins thus recovered were subjected to electrophoresis on polyacrylamide gel and the dried gels placed with X-OMAT film with intensifying screens at −70°C for 7.5 h. Lanes A–C: Sequential immuno-precipitation with mAb 4C.2 followed by final immunoprecipitation with mAb M1/9.3HL (Lane D). Lanes E–G: Sequential immunoprecipitation with mAb M1/9.3HL, followed by final immuno-precipitation with mAb 4C.2 (Lane H).
4. Discussion The common leukocyte antigen (CD45) is a transmembrane glycoprotein found only on hematopoietic cells. It is structurally heterogenous, consisting of a family of isoforms ranging in Mr from 180000 to 220000 that are distributed in characteristic patterns within the hematopoietic systems: the 220000 isoform (CD45RA) is expressed by B-cells and the 180000 isoform by cortical thymocytes (CD45RO). Different patterns of isoforms are expressed by T-cells that appear to correlate with function and prior stimulation with antigens or mitogens. All eight possible isoforms of CD45 generated by the alternative splicing of exons 4, 5 and 6 have been detected at the level of mRNA and alternative splicing of exon 7 may also occur [34,35]. The new mAb, 4C and its subclones 4C.1 and 4C.2, described in this paper are directed against a trypsinsensitive epitope on the CD45 molecule. The distribution of the epitope on non-B, non-T, pre-B and B lymphoid cells, a subpopulation on monocytes and one of the T-cell lines, but not on other T-cell or macrophage cell lines or macrophages, suggests that the epitope recognized by 4C is coded by exons 4, 5 and 6 of the CD45 gene and is absent from the RO isoform (180 kd) found on thymocytes and some T-cells [35]. The pattern of 4C epitope distribution on different hematopoietic cells is similar to some of the previously described mAbs against CD45RA, such as RA3-3A1/ 6.1 and RA3-2C2/1 [24–26]. However, anti-CD45RA mAbs RA-3A1/6.1 and RA3-2C2/1 do not inhibit the binding of moAb 4C.2 to L5178Y cells. Also, MoAb 4C2 inhibits binding of RA.3-2C2/1 but not that of RA.3A1/6.1 to L5178Y cells, suggesting that the epitope against which RA3.2C2.1 is directed is in close proximity but not identical to the epitope for MoAb 4C, or that 4C.2 causes a steric hindrance to the binding of mAb RA3-2C2/1 to its epitope. The extracellular portion of CD45 is rod-like in structure [36]. Alternatively spliced isoforms with exons 4, 5 and 6 exhibit an extension from this rod-like structure as determined by electron microscopy with low-angle rotatory shadowing [36]. Since it appears that the epitope recognized by 4C.2 may be coded by exons 4, 5 or 6, it is possible that the adhesion of L5178Y to MLT may take place on the extended structure of CD45. mAbs 4C.1 and 4C.2 significantly inhibited heterotypic adherence between a leukemic cell line L5178Y and a marrow stromal cell line (MLT), while the control isotype MoAb (anti-MEL-14) and anti-CD45 mAbs M1/9.3 HL, RA3-2C2/1 and RA3-3A/6.1 did not. The specificity of the effect of MoAb’s 4C.1/4C.2 on L5178Y cells is shown by lack of an effect on adherence of T-cell lines to MLT. The mechanism by which mAb 4C inhibits L5178Y adherence to MLT was investigated. CD45, a tyrosine
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Fig. 4. mAb 4C.2 and other anti-CD45 mAbs bind to different epitopes on CD45. A total of 106 L5178Y cells were suspended in 200 ml of PBS +1% BSA and treated with unlabelled mAb(s) 4C.2 (10 mg ml − 1), M1/9.3HL (10 mg ml − 1), RA3-2C2/1 (10 mg ml − 1), or RA-3A1/6.1 (10 mg ml − 1) for 1 h at room temperature followed by removal of excess MoAb and by treated with FITC labelled 4C.2 (1 mg) for 1 h, washed ×3 with 5 ml PBS +1% BSA, resuspended in 1 ml buffer and analyzed on a FACScan for immunofluorescence. (top row A – D) Note that T145 and T146 in the figure represent mAbs RA.3-2C2/1 and RA.3-3A1/6.1, respectively. Similarly, L5178Y cells were pretreated with 10 × concentration of the same unlabelled antibodies followed by staining with FITC labelled MoAb RA.3-2C2/1 (middle row E – H) or FITC labelled MoAb RA.3-3A1/6.1 (I – L). At 10 × concentration, unlabelled MoAbs 4C.2, RA.3-2C2/1 and RA.3-3A1/6.1 inhibited binding of the corresponding FITC-labelled MoAb (A, E, I). MoAb M1/9.3H did not inhibit binding of any of the three FITC-labelled MoAb used (D, H, L). At 10 × concentration, MoAbs RA.3-3A1/6.1 and RA3-2C2/1 did not affect binding of each other (G and K) or 4C.2 to the L5178Y cells (B and C). MoAb 4C.2 (10 ×) inhibited binding of FITC-labelled RA3-2C2/1 (F), but not of FITC labelled RA3-3A1/6.1 (J). Note: T145, mAbRA3-2C2/1; T146, mAbRA3-3A1/6.1.
phosphatase, can regulate both signal transduction and T- and B-cell proliferation induced by lymphocyte surface receptor molecules such as CD2, CD3 and CD28. This activity of CD45 is only evident when CD45 is in close physical association with CD2, CD3 and CD28 on the T-cell surface and CD19 or Bgp95 on B-cells [37]. CD45 is also essential for antigen-induced proliferative responses of T-cells and signaling via the B-cell antigen receptors [38 – 40]. In the absence of CD45,
activation of the phosphotidylinositol second messenger system and induction of tyrosine phosphorylation by activation of the T-cell receptor does not occur [41,42]. Sodium orthovanadate and H2O2 are known to block protein tyrosine phosphatase activity. Furthermore, certain phosphorylated proteins are known to persist after treatment with these agents [43,44]. CD45 has recently been shown to be involved in regulation of a tyrosine kinase-dependent adhesion pathway in human B-
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Fig. 5. 4C epitope is trypsin sensitive, while M1/9.3HL epitope is not trypsin sensitive. Untreated L5178Y cells or cells treated with trypsin-EDTA for 15 min were incubated with either FITC-labelled mAb 4C.2, M1/9.3HL or an irrelant isotype MoAb for 1 h. Cells were washed × 3 and analyzed on a FACScan. (A) Staining pattern with an irrelevant isotype and FITC-labelled mAb M1/9.3. (B) Staining pattern with an irrelevant isotype mAb and mAb 4C.2.
lymphocytes [45]. Two of 13 anti-CD45 mAbs used by Wagner et al. [45] decreased lymphocyte homotypic aggregation induced by multiple reagents, including MHC class I and class II, CD19, CD20, CD21, CD40 and Leu-13 cell-surface molecules. On the contrary, certain anti-CD45 mAb can induce homotypic aggrega-
tion of normal lymphoid cells via upregulation of CD11a [46]. Notably, MoAb 4C does not mediate adherence to MLT via CD11a since MLT cells lack CD54, the ligand for CD11a. mAb 4C described here occasionally induces a weak homotypic aggregation of L5178Y cells which can be disrupted by gentle pipet-
Fig. 6. Tyrosine phosphorylation in L5178Y cells. (A) Resting tyrosine phosphorylation over a 30 min period. (B) Time course of PMA induced phosphorylation. (C) Time course of PMA induced phosphorylation of L5178Y cells pretreated with 4C.1. Three major proteins of 120, 110, and 80 kDa are phosphorylated in response to PMA stimulation.
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ting. In all the experiments reported in this paper, the co-incubated L5178Y cells-MLT were examined under an inverted microscope at the end of the 2 h co-incubation and homotypic aggregation was not observed. No significant effect of MoAb 4C.2 on tyrosine phosphorylation activity was noted, suggesting that the inhibition of adhesion of L5178Y cells to MLT is not mediated by enzymatic activity of CD45. The second possibility is that CD45 acts as an adhesion protein leading to heterotypic adhesion of L5178Y cells to MSC. The external domain of CD45 is an extended rod and thus, could interact with surface molecules of other cells [36,47]. Indirect evidence suggests that the larger form of CD22 (i.e. CD22b) on B-cells binds to the CD45 RO (180 kd isoform) on T-cells and to CD75, a surface a-2-6 sialyltransferase on tonsillar B-cells and some B-cell lines [48]. More recently, it has been shown that adherence of a lymphoid cell line (MOLT-4) to immobilized CD45, CS-1 fragment of fibronectin, and possibly also VCAM-1, is mediated via a short tripeptide L –D–V present on all three proteins [49]. It is possible that a ligand for the LDV binding site of CD45 is present on the MLT cell surface. However, the purified soluble form of the exterior domain of CD45, a single 160 kDa glycoprotein, neither binds BW5147, 70Z/3 (pre-B) or BDL-1 (Ly-1+B lymphoma) cells or inhibit interactions of the endogenously expressed CD45 [50].
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In summary, we have immunized rats with L5178Y murine lymphoma cells and produced monoclonal antibodies through fusion of the rat spleen cells with nonsecretory murine myeloma cells. One of these antibodies, 4C and its subclones, were capable of inhibiting L5178Y adherence to MLT. This antibody recognized an epitope on the extended extracellular portion of murine CD45. This is the first report of adherence function of CD45 in interaction of marrow stromal cells and B-cell-lymphoma cells. No evidence was found for alteration of phosphatase activity of CD45 by these antibodies, suggesting that the inhibition of adherence was through interaction of the extracellular portion of CD45 with an unknown counterstructure on MLT cells. The probable location of the epitope recognized by 4C on the extended extracelluar portion of CD45 is consistent with such an adherence reaction. The distribution of CD45 isoforms suggests the possibility that adherence of lymphocytes subsets bearing the extended forms of CD45 may be differentially influenced by marrow stromal cells.
Acknowledgements We are grateful to Nancy Fernandez for typing the manuscript and Dr T. Van at the Automated Cytometry Laboratory M.D. Anderson Cancer Cancer Center for help with the immunofluorescence studies. This work was supported by US Public Health Service Grant CA47436 to Harinder S. Juneja, M.D., and AI 23521 to F.C. Schmalstieg. The automated cytometry laboratory at M.D. Anderson Cancer Center is supported by a grant from the National Cancer Institute (CA-16672).
References
Fig. 7. Densitometric analysis of the 80 kDa protein. Filled circles are resting L5178Y cells, filled squares are L5178Y cells stimulated with PMA, and filled triangles are L5178Y cells pretreated with 4C.1 and stimulated with PMA. While the kinetics of phosphorylation are similar, the 4C.1 treated cells demonstrate a slightly prolonged peak of phosphorylation and a modest decrease in phosphorylation relative to the untreated, PMA stimulated cells.
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[28] Miyake K, Medina K, Ishihara K, Kimoto M, Auenbach R, Kincade PW. A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture. J Cell Biol 1991;114:557. [29] Boue DR, Lebien TW. Structural characterization of the human B lymphocyte-restricted differentiation antigen CD22. Comparison with CD21 (complement receptor type 2/Epstein – Barr virus receptor). J Immunol 1988;140:192. [30] Hardy RR. Purification and characterization of monoclonal antibodies. In: Weir DM, editor. Immunochemistry. Boston, MA: Blackwell Scientific, 1986:13.11. [31] Juneja HS, Schmalstieg FC, Lee S, Chen J. Vascular cell adhesion molecule-1 and VLA-4 are obligatory adhesion proteins in the heterotypic adherence between human leukemia/lymphoma cells and marrow stromal cells. Exp Hematol 1993;21:444. [32] Goldman SJ, Uniyal S, Ferguson LM, Golan DE, Burakoff SJ, Kiener PA. Differential activation of phosphotyrosine protein phosphatase activity in a murine T-cell hybridoma by monoclonal antibodies to CD45. J Biol Chem 1992;267:6197. [33] Johnson P, Greenbaum L, Bottomly K, Trowbridge IS. Identification of the alternatively spliced exons of murine CD45 (T200) required for reactivity with B220 and other T200-restricted antibodies. J Exp Med 1989;169:1179. [34] Ralph SJ, Thomas ML, Morton CC, Trowbridge IS. Structural variation of human T200 glycoprotein (leukocyte common antigen). EMBO J 1987;6:1251. [35] Trowbridge IS. CD45. A prototype for membrane protein tyrosine phosphatases. J Biol Chem 1991;266:23517. [36] McCall MN, Shotton DM, &Barclay AN. Expression of soluble isoforms of rat CD45. Analysis by electron microscopy and use in epitope mapping of anti-CD45R monoclonal antibodies. Immunology 1992;76:310. [37] Ledbetter JA, Tonks NK, Fischer EH, Clark EA. CD45 regulates signal transduction and lymphocyte activation by specific association with receptor molecules on T- or B-cells. Proc Natl Acad Sci 1988;85:8628. [38] Pingel JT, Thomas ML. Evidence that the leukocyte-common antigen is required for antigen-induced T lymphocyte proliferation. Cell 1989;58:1055. [39] Weaver CT, Pingel JT, Nelson JO, Thomas ML. CD8 + T-cell clones deficient in the expression of the CD45 protein tyrosine phosphatase have impaired responses to T-cell receptor stimuli. Mol Cell Biol 1991;11:4415. [40] Justement LB, Campbell KS, Chien NC, Cambier JC. Regulation of B-cell antigen receptor signal transduction and phosphorylation by CD45. Science 1991;252:1839. [41] Koretzky GA, Picus J, Thomas ML, Weiss A. Tyrosine phosphatase CD45 is essential for coupling T-cell antigen receptor to the phosphatidyl inositol pathway. Nature 1990;346:66. [42] Koretzky GA, Picus J, Schultz T, Weiss A. Tyrosine phosphatase CD45 is required for T-cell antigen receptor and CD2-mediated activation of a protein tyrosine kinase and interleukin 2 production. Proc Natl Acad Sci USA 1991;88:2037. [43] Swarup GS, Cohen DL. Inhibition of membrane phosphotyrosylprotein phosphatase activity by vanadate. Biochem Biophys Res Commun 1982;107:1104. [44] Heffetz D, Bushkin I, Dror R, Zick Y. The insulinmimetic agents H2O2 and vanadate stimulate protein tyrosine phosphorylation in intact cells. J Biol Chem 1990;265:2896. [45] Wagner N, Engel P, Tedder TF. Regulation of the tyrosine kinase-dependent adhesion pathway in human lymphocytes through CD45. J Immunol 1993;150:4887. [46] Jack AS, Chapman KJ. Adhesion of normal and neoplastic lymphoid cells to fibroblasts. J Pathol 1991;164:151. [47] Woollett GR, Williams AF, Shotton DM. Visualization by low-angle shadowing of the leucocyte-common antigen. A major cell surface glycoprotein of lymphocytes. EMBO J 1985;4:2827.
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CD45 partially mediates heterotypic adhesion between murine leukemia/lymphoma cell line L5178Y and marrow stromal cells Harinder S. Juneja a,*, Frank C. Schmalstieg b, Sang Lee a, Jian Chen a a
Department of Internal Medicine, Di6ision of Hematology/Oncology, Uni6ersity of Texas Health Science Center, Room 5.016 MSB, 6431 Fannin Street, Houston, TX 77030, USA b The Department of Pediatrics, Uni6ersity of Texas, Medical Branch, Gal6eston, Gal6eston, TX, USA Received 17 November 1997; accepted 6 March 1998
Abstract We raised mAbs to whole L5178Y leukemia/lymphoma (LL) cells to identify adhesion proteins involved in adherence between LL cells and marrow stromal cells. One mAb, 4C, and its subclones 4C.1 and 4C.2 inhibited adherence of L5178Y LL cells to MLT, a nontransformed murine marrow stromal cell line. These MoAbs are directed against CD45RA. Control anti-CD45 mAbs and isotype mAbs were non-inhibitory. Other anti-CD45 mAbs, M1/9.3, RA3-3A1/6.1 and RA3-2C2/1 do not compete with mAb 4C.1 for binding to the L5178Y cell surface, but mAb 4C.1 competes for binding of mAb RA3-2C2/1. Effects of mAb 4C on tyrosine-phosphatase activity of CD45 in L5178Y cells are minimal, suggesting direct involvement of CD45 as an adhesion protein. © 1998 Elsevier Science Ltd. All rights reserved. Keywords: CD45; Marrow stromal cells; Adhesion; Leukemia; Lymphoma; Metastasis; Cell adhesion; Lymphocytes; Integrins
1. Introduction Marrow stromal cells are essential for sustained hematopoiesis both in vivo and in vitro [1 – 6]. In addition, factor-dependent hematopoietic cell lines can become factor independent when co-cultured in vitro with marrow stromal cells for several weeks to months [7 – 11]. During normal hematopoiesis in long-term cultures and the development of leukemic changes in the factor-dependent cell lines, the hematopoietic cells are in close apposition or adherent to the marrow stromal cells [9,12–14]. Physical apposition of pre-B-cells and marrow stromal cells is also essential for long-term B-cell lymphopoiesis in vitro [3,15]. We have previously utilized two murine leukemia/lymphoma (LL) cell lines, L5178Y and L1210, and a nontransformed murine marrow stromal cell line (MLT) to dissect the mechanism of LL cell adherence to marrow stromal cells [16]. MLT is a unique cell line that lacks expression of the
* Corresponding author. 0145-2126/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved. PII: S0145-2126(98)00070-8
intercellular adhesion molecule, ICAM-1. Studies with inhibitory monoclonal antibodies (mAbs) indicated that CD44, CD11a and b, CD18 and CD62 are not involved in LL cell adherence to MLT. Neoglycoprotein probes, mannosyl-bovine serum albumin (BSA) and galactosylBSA caused a partial, but significant, inhibition of LL cell adherence to MLT. In contrast, fucosyl-BSA had no inhibitory effects in this system. These data suggested that: (i) adherence of LL cells to marrow stromal cells involves a lectin mechanism with mannosyl and galactosyl specificities; and (ii) other mechanisms of adherence, not yet described, were also important in this system. To determine the role of as yet undescribed adhesion proteins, we raised rat mAbs to whole L5178Y LL cells. We present data on characterization of one mAb, 4C and its subclones, 4C.1 and 4C.2, which inhibited adherence of L5178Y LL cells to MLT. mAbs PS/2 (anti-CD49d) and MK/2 (anti-CD106) were also utilized to determine the role of integrin VLA-4 and its ligand vascular cell adhesion molecule-1 (VCAM-1), in this system.
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2. Materials and methods
2.1. Marrow stromal cell line A previously described murine marrow stromal cell line (MLT) was utilized. These marrow stromal cells are fibroblast-like in appearance, peroxidase and leukocyte alkaline phosphatase negative and non-specific esterase positive. Importantly, the MLT cell line does not express intercellular adhesion molecule-1 [16]. It was derived from long-term cultures of marrow obtained from DBA/2J mice and is passaged in vitro bi-weekly in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% fetal calf serum. The MLT cells were grown to confluence in 24-well plates for the adherence experiments.
2.2. Leukemia/lymphoma cell lines L5178Y, a lymphoma cell line originally developed from methylcholanthrene-treated DBA strain mice, was obtained from the NCI (DCT tumor repository, Frederick, MD). In our hands, the L5178Y cell line obtained from the repository stains positive with mAb 14.8 (TIB 164-ATCC, Rockville, MD) [17] which reacts with an epitope on B220 glycoprotein expressed by murine B-cells, B-cell precursors and a subpopulation of peripheral T-cells. The L5178Y cells are passaged intraperitoneally in DBA/2J mice (Harlan Sprague– Dawley, Houston, TX). At monthly intervals, fresh in vitro cultures of L5178Y cells are established from in vivo passaged cells in an attempt to maintain a constant phenotype. Several pre-B-, B-, T-cell and monocyte macrophage cell lines, listed in Table 1, were used in Table 1 Distribution of 4C epitope on myeloid and lymphoid cell lines Cell lines
Type
4C.1
M1/9.3HL
P388 L1210 70Z A20 L5178Y S49.1 S49 TIM.4 CTLL-2 P1793 Wehi 274-1 Wehi 3 IC-21
Lymphoblast Early lymphoblast Pre-B B-cell B-cell T Thy 1.2 T Thy-1− T TL(−) T T Monocyte Mono-Macro Macrophage
70/4 1000/11 300/10 16/1 300/15 8/6 9/6 13/5 20/20 6/3 30/3 3/3 5/3
ND 400/5 ND ND 1500/15 200/6 700/6 130/5 ND 600/3 120/3 130/3 150/3
Several cell lines were examined for expression of the M1/9.3HL and 4C epitopes using indirect immunofluorescence techniques. The stained cells were analyzed on a FACScan. The table shows the mean fluorescence channel under test/control conditions. Controls represent staining with only the FITC-labelled goat anti-rat antibody. All cell lines were obtained from ATCC (Rockville, MD). ND, not done.
characterization of the MoAb 4C. A set of well characterized parental CD45 + , mutant CD45 − , and revertant CD45 + T-cell lymphoma lines (BW5147G.1.4OUAR.1, BW5147 (T200 − a) 5.1 and BW5147 (Rev)1.1, respectively) were kindly provided by Dr Robert Hyman (Salk Institute, La Jolla, CA) [18]. These lymphoma cell lines were also grown in IMDM supplemented with 10% FCS.
2.3. Production of mAb against L5178Y cells Intact L5178Y cells were injected i.p. into 12–16 week old Wistar–Furth rats (Jackson Laboratories, Bar Harbor, ME). Hybridomas were generated by fusing nonsecretory myeloma cells (P3× 63Ag8.653) (ATCC, Rockville, MD) with spleen cells from the immunized rats, using standard protocols [19,20]. The hybridomas were first screened for reactivity against cell surface antigens on L5178Y cells by a cell-ELISA technique [20,21]. Conditioned medium, from clones positive on the ELISA screen, was used in an adherence assay to select hybridomas that inhibited adherence of L5178Y cells to MLT monolayers. A single mAb 4C and its subclones, 4C.1 and 4C.2, were selected for characterization (see below).
2.4. Heterotypic adherence between L5178Y and T-lymphoma cell lines and MLT Adherence of 51chromium (Cr) labelled L5178Y cells or T lymphoma cell lines to MLT monolayers was determined using previously described methodology [22]. Briefly, varying numbers of radiolabelled L5178Y cells or T lymphoma cells were added to MLT monolayers grown in IMDM and 10% FCS. After a 2 h incubation at 37°C in the presence of 75% N2, 20% O2 and 5% CO2, the wells were washed gently with PBS (pH 7.2) three times, and the adherent cells were lysed with 1N sodium hydroxide. The radioactivity recovered from the adherent layer was used to calculate the LL cell adherence to MLT. The quantitative adherence of L5178Y LL cells to MLT cells has not changed over a period of 4 years.
2.5. Effect of mAbs on heterotypic adherence of L5178Y cells to MLT L5178Y cell adherence to MLT was determined as described above in the presence or absence of a test mAb. mAbs used were 4C.1, M1/9.3HL (anti-T200) and anti-CD45RA mAb’s RA3-2C2/1 and RA3-3A1/ 6.1 [20,23–25]. mAb M1/9.3HL, directed against an epitope expressed by all lines of leukocyte differentiation, was either obtained from Boehringer Mannheim (Indianapolis, IN) or prepared from a hybridoma (TIB122) obtained from ATCC. F(ab)2 fragment of
H.S. Juneja et al. / Leukemia Research 22 (1998) 805–815
mAb 4C.1 was prepared by previously described methods [26]. Monoclonal isotype immunoglobulins (Chemicon, Temecula, CA) were used as isotype controls. Sodium bi-sulphate precipitated and dialyzed Ig form of mAbs PS/2 and MK/2 (ATCC Rockville, MD); [27,28] were utilized at a concentration of 10 mg ml − 1 in similar experiments to determine the role of integrin CD49d and its ligand, CD106, in our system. The adherence of L5178Y cells to MLT under the test conditions is represented as percent of controls. Percent adherence is presented as the mean (9 1 S.D.). All experiments were done in triplicate and repeated 3–21 times. Effect of mAb 4C.1 on heterotypic adherence L5178Y cells and T-cell lymphoma cell lines (BW5147G.1.4OUAR.1, BW5147 (T200 − a) and BW5147 (Rev)) to MLT was also determined in concurrent experiments.
2.6. Cell surface iodination of L5178Y cells L5178Y cells (2× 107) were suspended in 1 ml of PBS, pH 7.4 and placed on ice. All subsequent steps were performed on ice. A total of 5 U of lactoperoxidase and 5 mCi of 125I were then added followed by 20 ml of 0.06% H2O2. After 5 min, 10 ml of 0.06% H2O2 was added and the incubation was carried out for an additional 5 min. The cells were washed three times and subsequently lysed in 150 ml of buffer containing 0.5% NP-40, 5 mM PO4, 150 mM NaCl, 10 mM pyrophosphate, 25 mM NaF, 1 mM ZnC12, 5 mM EDTA, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 10 mg ml − 1 each of soybean trypsin inhibitor, anti-papain, leupeptin, and pepstatin A, and 20 mM HEPES, pH 7.4 [29]. The cell lysate was centrifuged for 1 min to remove DNA. The supernatant was removed and saved.
2.7. Immunoprecipitation of L5178Y cell membrane lysate by mAbs, 4C.2 and M1 /9.3HL Affinity purified mAbs 4C.2 and M1/9.3HL were prepared by using a commercial kit (ImmunoPure Ag/ Ab Immobilization Kit, Pierce, Rockford, IL). The lysed membrane preparation was precleared as follows: Irrelevant 50 ml of nonimmune rabbit serum was added to each supernatant and the sample incubated with rocking overnight at 4°C. After the preclearing procedure, the radio-labelled cell lysate was aliquoted into two portions and 1 mg of 4C.2 or M1/9.3HL antibodies, previously bound to 0.2 ml suspension of protein G sepharose A beads (2.4 mg IgG binding capacity) (Sigma), was added to the aliquots. Incubation was carried out for 30 min at 4°C. These incubations were repeated four times and then the alternate antibody was added to each supernatant. The protein G sepharose
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beads containing the immune complexes were washed 5× with PBS. Sample buffer (15 ml, 2.3% SDS and 0.36 M 2-ME) was added to the beads, without boiling. The iodinated proteins thus recovered from the beads were subjected to electrophoresis on 10% polyacrylamide gels. After drying, the gels were placed with X-OMAT film (Eastman Kodak, Rochester, NY) with intensifying screens at − 70°C for 7.5 h.
2.8. Competiti6e binding studies In order to determine if other anti CD45 mAbs were directed against the same epitope on the CD45 molecule as 4C, competitive binding studies were performed in the following manner. Affinity purified mAbs were fluorescein labelled by standard techniques [30]. L5178Y cells were suspended in 200 ml of PBS+1% BSA and treated with 10 mg ml − 1 of unlabelled affinity purified mAbs M1/9.3HL, 4C.2, RA3-2C2/1 and RA3A1/6.1 for 1 h at room temperature followed by treatment with 1 mg of FITC labelled 4C.2 for 1 h. Cells were washed three times with 5 ml PBS+ 1% BSA and resuspended in 1 ml buffer and analyzed on a FACScan (Becten-Dickinson, Mountain View, CA) as described below. To determine further similarities or differences between the epitopes for mAbs 4C.2 and M1/9.3 HL, untreated L5178Y cells and L5178Y cells treated with 0.5% trypsin-EDTA (Sigma, St. Louis, MO) for 15 min were stained with FITC labelled 4C.2 or FITC labelled isotype Ig.
2.9. Expression of 4C.2 and M1 /9.3HL epitopes in hematopoietic tissues and on hematopoietic cell lines Peripheral blood and the hematopoietic tissues (bone marrow, spleen and thymus) were obtained from 8–10 week old DBA/2 mice and single cell suspensions prepared. Single cell suspensions from the tissues were prepared by cutting the tissues into small pieces, homogenization of the tissue in a syringe and rinsing out of cells released. The cell suspension was then allowed to stand for 5 min and the supernatant passed through a 27 gauge needle to remove large clumps and fibrous tissue. A panel of murine hematopoietic cell lines of myeloid, monocytic and lymphoid origin were obtained from ATCC (Table 1). A total of 106 cells from each of the tissues or the cell lines were suspended in PBS+1% BSA in a 2 ml microfuge tube and incubated with 4C.1, M1/9.3HL or an isotype mAb for 60 min followed by incubation with an FITC labelled goat anti-rat Ig antibody for 45 min. After washing the cells twice with 2 ml PBS+ 1% BSA, the cells were analyzed on a FACScan utilizing the LYSIS software (BD, CA). The analysis were performed in the Automated Cytometry core facility at the University of Texas M.D. Anderson Cancer Center as previously described [31].
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Fig. 1. mAbs 4C.1 and PS/2 inhibit L5178Y adherence to MLT. 51Cr-labelled L5178Y cells were incubated with each of the mAbs diluted in IMDM+ 2% BSA, separately, for 1 h and allowed to adhere to MLT monolayers in the presence of the same mAb for 2 h. At the end of the assay, the L5178Y cell viability, by dye-exclusion techniques, was ]95%. Also, no homotypic aggregation of L5178Y cells was noted under the inverted microscope. Adherence of L5178Y cells to MLT in presence of each mAb is presented as a percentage of the adherence of L5178Y cells to MLT under control conditions. * P B0.05 on a two-sample analysis by Student’s t-test (Statgraphics, Rockville, MD) on comparison with the controls. (A) Test mAbs included 4C.1 conditioned medium (undiluted) or ascites (1:100). F(ab)2 fragment of mAb4C.1 (10 mg ml − 1) and rat IgG 2a isotype control (MEL-14). Adhesion of L5178Y cells under control conditions (IMDM + 2% BSA) ranged from 13.6 95.5 to 22.5 9 12.4%. (B) Effect of other anti-CD45 mAbs, i.e. M1/9.3HL (1 ×conditioned medium), RA3-2C2/1 (Ig 10 mg ml − 1) and RA3A1/6.1 (Ig 10 mg ml − 1) and PS/2 (Ig 10 mg ml − 1) is represented as a percentage of isotype controls (rat IgM, rat IgG 2a and rat IgG 2b, respectively). Data with mAb MK/2 is represented as a percent of control with IMDM +2% BSA. Adhesion of L5178Y to MLT in the controls ranged from 18.9 9 6.8 to 24.2 9 2.4%. Isotype mAbs were used as Ig form (10 mg ml − 1). (n), number of observations.
2.10. Tyrosine phosphorylation in L5178Y cells L5178Y cells (1 × 107) were suspended in 500 ml of RPMI-1640 in each of 18 tubes. Anti-CD45 (4C.1) (14 mg) was added to certain tubes and incubated for 1 min at room temperature. PMA (0.5 mg) was added to all but the control samples, and the tubes brought to 37°C. At 0, 2, 5, 10, 20 and 30 min, 500 ml of ice-cold PBS containing 500 mM sodium orthovanadate was added and the cells washed once in this medium. Following the wash, the cells were pelleted and lysed in 150 ml of cold lysis buffer (1% NP-40, 5 mM PO4, 150 mM NaC1, 10 mM pyrophosphate, 1 mM sodium orthovanadate, 25 mM NaF, 1 mM ZnC12, 5 mM EDTA, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 10 mg ml − 1 each of soybean trypsin inhibitor, leupeptin, and pepstain A, and 20 mM HEPES, pH 7.4) for 20 min on ice. The lysates were centrifuged to remove nuclei and cell debris and immediately added to 2 ×SDS sample buffer (reducing) and boiled for 2 min. The samples were subjected to electrophoresis on 10% polyacrylamide gels, and after electrophoretic transfer to nitrocellulose, blocked in PBS containing 5% BSA, 0.05% azide, and 500 mM sodium orthovanadate for 4 h
at 4°C. The phosphotyrosine containing proteins were reacted with murine monoclonal anti-phosphotyrosine antibody (Sigma). The second antibody was rabbit anti-murine immunoglobulin conjugated to horseradish peroxidase. Detection was accomplished by addition of 0.025% H2O2 and 4-chloro-1-naphthol as previously described [32]. The images were digitalized and densitometric analyses performed.
3. Results Of the 250 clones that were positive on the ELISA screen, only one mAb, 4C, and its subclones 4C.1 and 4C.2, significantly inhibited adherence of L5178Y cells to MLT. Using an isotyping kit (Binding Site, San Diego, CA) mAb 4C and its subclones were identified as rat IgG2a proteins. Data from experiments with subclone 4C.1 is shown in Fig. 1. F(ab)2 fragments of 4C.1 also inhibited L5178Y adherence to MLT by 23.59 5.1% (Fig. 1). It is important to note that isotype-control (IgG2a) antibody, MEL-14, did not cause a decrease in L5178Y adherence to MLT. In contrast, anti-CD45 mAbs M1/9.3HL, RA3-2C2/1 and RA3-
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Fig. 2. Inhibitory activity of mAb 4C on heterotypic adherence between L5178Y and marrow stromal line (MLT) is specific. 51Cr labelled L5178Y and T lymphoma cell lines (BW5147G.1.4OUAR.1, BW5147 (T200 − a) and BW5147 (Rev)) were incubated in 4C.1CM (1 × ) for 1 h and then allowed to adhere to MLT monolayers for 2 h in the continuous presence of mAb 4C.1. At end of 2 h, the percent of adherence LL cells was determined as described in Section 2. Adherence is represented as the percent of the total number of cells (input) added to the MLT monolayer. The T-cell lymphoma cell lines show a greater adherence to MLT than L5178Y cells. Adherence of L5178Y to MLT is significantly inhibited by mAb 4C while the adherence of the T lymphoma cells to MLT was unaffected.
3A1/6-1 did not inhibit the adherence of L5178Y cells to MLT. mAb PS/2 (anti-CD49d) caused a significant reduction, while mAb MK/2 (anti-CD106) caused an increase in L5178Y adherence to MLT (Fig. 1). Notably, mAb 4C did not inhibit the adherence of the CD45 + , mutant and revertant T lymphoma cell lines to MLT, suggesting that its effect on L5178Y-MLT interaction is not nonspecific, but rather, a specific effect (Fig. 2).
3.1. Comparison of 4C and M1 /9.3HL Radio-iodination of L5178Y cells was carried out to compare the antigen specificities of mAbs 4C.2 and M1/9.3HL (Fig. 3). Sequential clearing of radio-labelled cell lysates were performed with each antibody and final immunoprecipitation attempted with the alternative antibody. Both antibodies recognized the same 230 – 240 kDa protein, establishing 4C as an anti-CD45 antibody.
3.2. Competiti6e binding to the CD45 on the cell surface by 4C and other known anti-CD45 mAbs Competition experiments demonstrated that mAbs M1/9.3HL, RA3-2C2/1 and RA3-3A1/6.1 do not compete for binding of FITC-labelled mAb 4C.2 to L5178Y cells. Unlabelled 4C.2, at 10× concentration, competes
with binding of FITC labelled 4C.2 (Fig. 4, top row). MoAb 4C.2 effectively competed with binding of FITC labelled RA3-2C2/1 (Fig. 4, middle row) but not with FITC labelled RA3-3A1/6.1 (Fig. 4 bottom row). MoAb RA3-2C2/1 is directed against an epitope dependent on the expression of the first alternatively spliced exon (exon A) of the CD45 molecule and expressed on B-cells and at a lower level on CD8 + T-cells, while MoAb RA3-3A1/6.1 is also directed against the RA isoform of CD45 but the exact epitope has not been identified [33]. MoAb M1/9.3HL did not compete with 4C.2, RA3-2C2/1 or RA3-A1/6.1. mAb 4C is directed against a trypsin-sensitive epitope on CD45, while M1/ 9.3HL is directed against a trypsin-resistant epitope on CD45 (Fig. 5). Expression of the M1/9.3HL and 4C epitopes on hematopoietic cell lines is shown in Table 1. The M1/ 9.3HL epitope was present on pre-B-, pro B-, B-, Tand monocyte-macrophage cell lines. In contrast, the 4C epitope was present on pre-B, B and non-B-non-T lymphoid cells but not on the majority of the macrophages-monocytes or T-cells. Only one T-cell line, TIMI-4, was very weakly positive on FACScan analysis and showed a patchy membrane staining pattern on indirect immunofluorescence microscopy. Peripheral blood lymphocytes stained strongly positive with mAb M1/9.3HL, while monocytes and neutrophils had two subgroups, one weak and the other strongly
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positive. In contrast, staining with mAb 4C.2 revealed two lymphocyte subpopulations, one negative and the other positive. A vast majority (#85%) of granulocytes and monocytes did not exhibit the 4C epitope. A small percentage of monocytes may be positive, which is in keeping with expression of 4C epitope on some of the monocyte-macrophage cell lines. This data is consistent with the distribution of the 220 kd isoform of CD45. A single peak of M1/9.3HL positive cells was observed on cells isolated from the bone marrow and spleen. Based on the 4C epitope expression, both the spleen and the bone marrow cells could be divided into a positive and a negative subpopulation. Lymphoid cells of the thymus were negative for 4C, but M1/9.3HL positive. Thus, 4C and M1/9.3HL epitopes are distributed differentially on hematopoietic cells (data not shown).
3.3. Influence of 4C.1 on tyrosine phosphorylation in L5178Y cells In order to determine whether 4C had an effect on tyrosine phosphorylation, and hence cell signaling in L5178Y cells, kinetics of PMA-induced phosphorylation with and without 4C.1 was carried out. PMA induced an immediate increase in phosphorylation of 130, 110, and 80 kDa proteins in L5187Y cells (Fig. 6). 4C.1 had little effect on the kinetics of phosphorylation of the 80 kDa protein (Fig. 7).
Fig. 3. mAb 4C.2 immunoprecipitates the same protein as mAb M1/9.3HL, i.e. CD45 from L5178Y cell membrane. Membrane proteins of L5178Y cells were iodinated with 125I. The membrane was lysed and precleared with nonimmune rabbit serum. Aliquots of the cell membane lysate were treated with protein G-Sepharose A bead bound mAbs 4C.2 or M1/9.3HL. These incubations were repeated × 4 and then alternate antibody was added to each supernatant. The protein G-sepharose beads containing the immune complexes were washed repeatedly with PBS. The immune complexes were removed by treatment with SDS/2-ME buffer, without boiling. The iodinated proteins thus recovered were subjected to electrophoresis on polyacrylamide gel and the dried gels placed with X-OMAT film with intensifying screens at −70°C for 7.5 h. Lanes A–C: Sequential immuno-precipitation with mAb 4C.2 followed by final immunoprecipitation with mAb M1/9.3HL (Lane D). Lanes E–G: Sequential immunoprecipitation with mAb M1/9.3HL, followed by final immuno-precipitation with mAb 4C.2 (Lane H).
4. Discussion The common leukocyte antigen (CD45) is a transmembrane glycoprotein found only on hematopoietic cells. It is structurally heterogenous, consisting of a family of isoforms ranging in Mr from 180000 to 220000 that are distributed in characteristic patterns within the hematopoietic systems: the 220000 isoform (CD45RA) is expressed by B-cells and the 180000 isoform by cortical thymocytes (CD45RO). Different patterns of isoforms are expressed by T-cells that appear to correlate with function and prior stimulation with antigens or mitogens. All eight possible isoforms of CD45 generated by the alternative splicing of exons 4, 5 and 6 have been detected at the level of mRNA and alternative splicing of exon 7 may also occur [34,35]. The new mAb, 4C and its subclones 4C.1 and 4C.2, described in this paper are directed against a trypsinsensitive epitope on the CD45 molecule. The distribution of the epitope on non-B, non-T, pre-B and B lymphoid cells, a subpopulation on monocytes and one of the T-cell lines, but not on other T-cell or macrophage cell lines or macrophages, suggests that the epitope recognized by 4C is coded by exons 4, 5 and 6 of the CD45 gene and is absent from the RO isoform (180 kd) found on thymocytes and some T-cells [35]. The pattern of 4C epitope distribution on different hematopoietic cells is similar to some of the previously described mAbs against CD45RA, such as RA3-3A1/ 6.1 and RA3-2C2/1 [24–26]. However, anti-CD45RA mAbs RA-3A1/6.1 and RA3-2C2/1 do not inhibit the binding of moAb 4C.2 to L5178Y cells. Also, MoAb 4C2 inhibits binding of RA.3-2C2/1 but not that of RA.3A1/6.1 to L5178Y cells, suggesting that the epitope against which RA3.2C2.1 is directed is in close proximity but not identical to the epitope for MoAb 4C, or that 4C.2 causes a steric hindrance to the binding of mAb RA3-2C2/1 to its epitope. The extracellular portion of CD45 is rod-like in structure [36]. Alternatively spliced isoforms with exons 4, 5 and 6 exhibit an extension from this rod-like structure as determined by electron microscopy with low-angle rotatory shadowing [36]. Since it appears that the epitope recognized by 4C.2 may be coded by exons 4, 5 or 6, it is possible that the adhesion of L5178Y to MLT may take place on the extended structure of CD45. mAbs 4C.1 and 4C.2 significantly inhibited heterotypic adherence between a leukemic cell line L5178Y and a marrow stromal cell line (MLT), while the control isotype MoAb (anti-MEL-14) and anti-CD45 mAbs M1/9.3 HL, RA3-2C2/1 and RA3-3A/6.1 did not. The specificity of the effect of MoAb’s 4C.1/4C.2 on L5178Y cells is shown by lack of an effect on adherence of T-cell lines to MLT. The mechanism by which mAb 4C inhibits L5178Y adherence to MLT was investigated. CD45, a tyrosine
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Fig. 4. mAb 4C.2 and other anti-CD45 mAbs bind to different epitopes on CD45. A total of 106 L5178Y cells were suspended in 200 ml of PBS +1% BSA and treated with unlabelled mAb(s) 4C.2 (10 mg ml − 1), M1/9.3HL (10 mg ml − 1), RA3-2C2/1 (10 mg ml − 1), or RA-3A1/6.1 (10 mg ml − 1) for 1 h at room temperature followed by removal of excess MoAb and by treated with FITC labelled 4C.2 (1 mg) for 1 h, washed ×3 with 5 ml PBS +1% BSA, resuspended in 1 ml buffer and analyzed on a FACScan for immunofluorescence. (top row A – D) Note that T145 and T146 in the figure represent mAbs RA.3-2C2/1 and RA.3-3A1/6.1, respectively. Similarly, L5178Y cells were pretreated with 10 × concentration of the same unlabelled antibodies followed by staining with FITC labelled MoAb RA.3-2C2/1 (middle row E – H) or FITC labelled MoAb RA.3-3A1/6.1 (I – L). At 10 × concentration, unlabelled MoAbs 4C.2, RA.3-2C2/1 and RA.3-3A1/6.1 inhibited binding of the corresponding FITC-labelled MoAb (A, E, I). MoAb M1/9.3H did not inhibit binding of any of the three FITC-labelled MoAb used (D, H, L). At 10 × concentration, MoAbs RA.3-3A1/6.1 and RA3-2C2/1 did not affect binding of each other (G and K) or 4C.2 to the L5178Y cells (B and C). MoAb 4C.2 (10 ×) inhibited binding of FITC-labelled RA3-2C2/1 (F), but not of FITC labelled RA3-3A1/6.1 (J). Note: T145, mAbRA3-2C2/1; T146, mAbRA3-3A1/6.1.
phosphatase, can regulate both signal transduction and T- and B-cell proliferation induced by lymphocyte surface receptor molecules such as CD2, CD3 and CD28. This activity of CD45 is only evident when CD45 is in close physical association with CD2, CD3 and CD28 on the T-cell surface and CD19 or Bgp95 on B-cells [37]. CD45 is also essential for antigen-induced proliferative responses of T-cells and signaling via the B-cell antigen receptors [38 – 40]. In the absence of CD45,
activation of the phosphotidylinositol second messenger system and induction of tyrosine phosphorylation by activation of the T-cell receptor does not occur [41,42]. Sodium orthovanadate and H2O2 are known to block protein tyrosine phosphatase activity. Furthermore, certain phosphorylated proteins are known to persist after treatment with these agents [43,44]. CD45 has recently been shown to be involved in regulation of a tyrosine kinase-dependent adhesion pathway in human B-
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Fig. 5. 4C epitope is trypsin sensitive, while M1/9.3HL epitope is not trypsin sensitive. Untreated L5178Y cells or cells treated with trypsin-EDTA for 15 min were incubated with either FITC-labelled mAb 4C.2, M1/9.3HL or an irrelant isotype MoAb for 1 h. Cells were washed × 3 and analyzed on a FACScan. (A) Staining pattern with an irrelevant isotype and FITC-labelled mAb M1/9.3. (B) Staining pattern with an irrelevant isotype mAb and mAb 4C.2.
lymphocytes [45]. Two of 13 anti-CD45 mAbs used by Wagner et al. [45] decreased lymphocyte homotypic aggregation induced by multiple reagents, including MHC class I and class II, CD19, CD20, CD21, CD40 and Leu-13 cell-surface molecules. On the contrary, certain anti-CD45 mAb can induce homotypic aggrega-
tion of normal lymphoid cells via upregulation of CD11a [46]. Notably, MoAb 4C does not mediate adherence to MLT via CD11a since MLT cells lack CD54, the ligand for CD11a. mAb 4C described here occasionally induces a weak homotypic aggregation of L5178Y cells which can be disrupted by gentle pipet-
Fig. 6. Tyrosine phosphorylation in L5178Y cells. (A) Resting tyrosine phosphorylation over a 30 min period. (B) Time course of PMA induced phosphorylation. (C) Time course of PMA induced phosphorylation of L5178Y cells pretreated with 4C.1. Three major proteins of 120, 110, and 80 kDa are phosphorylated in response to PMA stimulation.
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ting. In all the experiments reported in this paper, the co-incubated L5178Y cells-MLT were examined under an inverted microscope at the end of the 2 h co-incubation and homotypic aggregation was not observed. No significant effect of MoAb 4C.2 on tyrosine phosphorylation activity was noted, suggesting that the inhibition of adhesion of L5178Y cells to MLT is not mediated by enzymatic activity of CD45. The second possibility is that CD45 acts as an adhesion protein leading to heterotypic adhesion of L5178Y cells to MSC. The external domain of CD45 is an extended rod and thus, could interact with surface molecules of other cells [36,47]. Indirect evidence suggests that the larger form of CD22 (i.e. CD22b) on B-cells binds to the CD45 RO (180 kd isoform) on T-cells and to CD75, a surface a-2-6 sialyltransferase on tonsillar B-cells and some B-cell lines [48]. More recently, it has been shown that adherence of a lymphoid cell line (MOLT-4) to immobilized CD45, CS-1 fragment of fibronectin, and possibly also VCAM-1, is mediated via a short tripeptide L –D–V present on all three proteins [49]. It is possible that a ligand for the LDV binding site of CD45 is present on the MLT cell surface. However, the purified soluble form of the exterior domain of CD45, a single 160 kDa glycoprotein, neither binds BW5147, 70Z/3 (pre-B) or BDL-1 (Ly-1+B lymphoma) cells or inhibit interactions of the endogenously expressed CD45 [50].
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In summary, we have immunized rats with L5178Y murine lymphoma cells and produced monoclonal antibodies through fusion of the rat spleen cells with nonsecretory murine myeloma cells. One of these antibodies, 4C and its subclones, were capable of inhibiting L5178Y adherence to MLT. This antibody recognized an epitope on the extended extracellular portion of murine CD45. This is the first report of adherence function of CD45 in interaction of marrow stromal cells and B-cell-lymphoma cells. No evidence was found for alteration of phosphatase activity of CD45 by these antibodies, suggesting that the inhibition of adherence was through interaction of the extracellular portion of CD45 with an unknown counterstructure on MLT cells. The probable location of the epitope recognized by 4C on the extended extracelluar portion of CD45 is consistent with such an adherence reaction. The distribution of CD45 isoforms suggests the possibility that adherence of lymphocytes subsets bearing the extended forms of CD45 may be differentially influenced by marrow stromal cells.
Acknowledgements We are grateful to Nancy Fernandez for typing the manuscript and Dr T. Van at the Automated Cytometry Laboratory M.D. Anderson Cancer Cancer Center for help with the immunofluorescence studies. This work was supported by US Public Health Service Grant CA47436 to Harinder S. Juneja, M.D., and AI 23521 to F.C. Schmalstieg. The automated cytometry laboratory at M.D. Anderson Cancer Center is supported by a grant from the National Cancer Institute (CA-16672).
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Fig. 7. Densitometric analysis of the 80 kDa protein. Filled circles are resting L5178Y cells, filled squares are L5178Y cells stimulated with PMA, and filled triangles are L5178Y cells pretreated with 4C.1 and stimulated with PMA. While the kinetics of phosphorylation are similar, the 4C.1 treated cells demonstrate a slightly prolonged peak of phosphorylation and a modest decrease in phosphorylation relative to the untreated, PMA stimulated cells.
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