Monoclonal antibodies to lymphocyte surface antigens for cetacean homologues to CD2, CD19 and CD21

Veterinary Immunology and Immunopathology 84 (2002) 209±221

Monoclonal antibodies to lymphocyte surface antigens for cetacean homologues to CD2, CD19 and CD21 Sylvain De Guise*, Karen Erickson, Myra Blanchard, Lisa DiMolfetto, Heather D. Lepper, Janice Wang, Jeffrey L. Stott, David A. Ferrick Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA Received 20 March 2000; received in revised form 8 August 2001; accepted 19 October 2001

Abstract CD2 is a pan-T cell marker, while CD19 and CD21 are important molecules in signal transduction of B lymphocytes. CD19 and CD21 are both present on mature B cells, while CD19 is also present in developing B cells and plasma cells. Monoclonal antibodies (mAbs) against cetacean lymphocyte putative homologues to CD2 (two different antibodies), CD19 and CD21 were characterized. The proteins immunoprecipitated were as follows: F21.I (putative anti-CD2), 43 and 59 kDa; F21.B (putative anti-CD19), 83 and 127 kDa; F21.F (putative anti-CD21), 144 kDa. The second putative anti-CD2 (F21.C) selectively inhibited the binding of F21.I. Both the putative antiCD2 (T cell markers) stained T-cell zones on lymph node sections, while both the B cell markers (putative CD19 and CD21) stained B-cell zones. F21.B and F21.F were absent from thymus single cell suspension but labeled 63 and 65% mesenteric lymph node lymphocytes, respectively, while both F21.C and F21.F were present on 100% thymocytes and fewer lymph node lymphocytes. B and T cell markers were mutually exclusive on double labeling using ¯ow cytometry. These mAbs are foreseen as possible valuable diagnostic and research tools to assess immune functions of captive and wild cetaceans. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Immunology; Cetaceans; Phenotyping; CD2; CD19; CD21

* Corresponding author. Present address: Department of Pathobiology, University of Connecticut, 61 N Eagleville Road, U-89, Storrs, CT 06269, USA. Tel.: ‡1-860-486-0850; fax: ‡1-860-486-2594. E-mail address: [email protected] (S. De Guise).

0165-2427/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 1 ) 0 0 4 0 9 - 3

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1. Introduction Monoclonal antibodies (mAbs) which identify subpopulations of peripheral blood leucocytes are essential to investigation of the role of these cells in the pathogenesis and regulation of disease (Horejsi, 1991). Although cross-reactivity of mAbs between phylogenically distant species has sometimes been observed (Jacobson et al., 1993), a better understanding of immune function and possible eventual therapeutic use of those mAbs call for species speci®c or closely related species cross-reactive reagents. The effort devoted to the development of mAbs against leucocyte cell surface markers is best re¯ected by the numerous symposia recently held to characterize the advance in different domestic species (Hopkins et al., 1993; Howard and Naessens, 1993; SaalmuÈller et al., 1994; Lunn et al., 1996). CD2 was ®rst recognized for its ability to form ``rosettes'' with sheep red blood cells and served as the earliest marker of human T-cells (Moingeon et al., 1989). It is one of the ®rst antigens expressed following colonization of the thymus by T-cell precursors and is detectable on the cell surface prior to the TCR/CD3 complex (Strominger, 1989). CD2 is found on more than 95% of thymocytes and maintained on virtually all peripheral mature T cells (Moingeon et al., 1989), although not exclusively on T cells. CD2 is now recognized for its roles in cellular adhesion and signal transduction (Moingeon et al., 1989). CD19 and CD21 are important molecules in signal transduction, expressed mostly, although not exclusively, on B lymphocytes (Tedder et al., 1994). CD21, which is also known as complement receptor 2 (CR2), and CD19 are part of a complex that link the acquired immunity through B cell function to natural immunity through complement activity (Matsumoto et al., 1991; Fearon and Carter, 1995). As part of our current efforts to develop assays and reagents to study the immune system of marine mammals, the present paper reports on the characterization of four mAbs against surface proteins of cetacean leucocytes developed in our laboratory. These putative homologues to CD2, CD19 and CD21 are foreseen as possible valuable diagnostic and research tools to assess immune functions of captive and wild cetaceans as part of the evaluation of their health status. 2. Material and methods 2.1. Antibodies used for characterization The antibodies used in this study include the following: isotype-speci®c, ¯uorescein isothiocyanate (FITC)-conjugated reagents (Zymed Laboratories, South San Francisco, CA); FITC-conjugated goat F(ab0 )2 anti-mouse (anti-Ms) IgG …H ‡ L† (Caltag, South San Francisco, CA); R-phycoerythrin (PE)-conjugated goat F(ab0 )2 anti-MS IgG …H ‡ L† (Molecular Probe, Eugene, OR); biotinylated horse anti-MS IgG (Vector Laboratories, Burlingame, CA); FITC-conjugated F(ab0 )2 goat anti-rabbit IgG …H ‡ L† (Caltag, South San Francisco, CA); rabbit polyclonal anti-killer whale Ig (Bernadette Taylor, University of California, Davis, CA); a control irrelevant antibody (B. cor.) (Myra Blanchard, University of California, Davis, CA).

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2.2. Production of mAbs BALB/c mice were immunized intraperitoneally at 3-week intervals with 2  106 peripheral blood mononuclear cells (PBMCs) from a healthy, captive bottlenose dolphin (Tursiops truncatus). mAbs were produced as previously described (BlanchardChannell et al., 1994), screened by ¯ow cytometry and cloned by limiting dilution. Antibody isotype was determined by ¯ow cytometry using isotype-speci®c, FITCconjugated reagents. Ascitic ¯uid was puri®ed using Bakerbond ABX mixed function ion-exchange matrix (J.T. Baker, Philipsburg, NJ). Puri®ed mAb were directly labeled with FITC (Bethyl Laboratories, Montgomery, TX) for later use in double labeling. 2.3. Cell and tissue preparation Cells for this study were from different sources. Blood samples from different species of healthy cetaceans kept in captivity as well as from beached animals in transit for rehabilitation purposes were received from Sea World Parks. Blood was kept cool and shipped overnight. Mesenteric lymph node, spleen and thymus were collected from a freshly dead wild common dolphin (Delphinus delphis) at San Pedro Marine Mammal Center (California), and a captive bottlenose dolphin who died in a Sea World Park. Shortly after death, the tissues were transferred to ice-cold RPMI 1640 with 10% fetal calf serum. Single cell suspensions from lymphoid organs were cryopreserved in fetal calf serum with 10% DMSO. For immunohistochemistry, lymphoid tissues were collected from relatively freshly dead stranded and/or captive cetaceans (Mote Marine Laboratory, Florida, and Naval Ocean System Center, California) and snap frozen in OCT Compound (Miles, Elkhart, IN) as in Blanchard-Channell et al. (1994). 2.4. Immunoprecipitations and PAGE Freshly isolated cells were used as antigen sources for immunoprecipitations. Leucocytes were surface-labeled with sulfo-NHS-biotin (Pierce, Rockford, IL) as previously described (Blanchard-Channell et al., 1994). Cellular proteins were solubilized with a lysis buffer (LB) containing 1.5% (w/v) Briji 99 and 0.5% Briji 96 (Blanchard-Channell et al., 1994). Puri®ed mAb were bound to agarose beads (af®gel-10 active ester agarose, Bio Rad, Richmond, CA) and incubated with labeled cell lysates overnight at 4 8C or for 2 h at room temperature. Bound proteins were removed from beads by incubation with sample application buffer (containing SDS and 2-mercaptoethanol), and analyzed by discontinuous SDS-polyacrylamide (PAGE; Laemmli, 1970), along with molecular weight standards. Proteins were transferred to Immobilon P membrane (Millipore, South San Francisco, CA) and after blocking (5% non-fat dry milk in Tris-buffered saline with 0.5% Tween-20), were probed with streptavidin-horseradish peroxidase (Zymed Laboratories, South San Francisco, CA). Membranes were developed with enhanced chemiluminescence (Amersham, Arlington Heights, IL) per the manufacturer's instructions and light emission recorded on hyper®lm-ECL (Amersham, Arlington Heights, IL).

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2.5. Immunofluorescence staining and flow cytometric analysis Cell preparations or whole blood samples were analyzed by ¯ow cytometry as previously described (Blanchard-Channell et al., 1994). Brie¯y, 1  106 leucocytes were blocked with 5% goat serum and indirectly stained with mAb using FITC-conjugated goat F(ab0 )2 anti-mouse (anti-Ms) IgG …H ‡ L† (Caltag, South San Francisco, CA). Nonspeci®c binding of MS Ig to cells was determined by indirect staining using an irrelevant antibody (B. cor.). Double labeling was used to simultaneously test for the binding of two different mAbs. Cells were ®rst incubated with one unlabeled mouse anti-cetacean mAb, followed by PE-labeled goat F(ab0 )2 anti-Ms, and ®nally incubated with a different FITCconjugated mouse anti-cetacean mAb. For double labeling with the rabbit anti-killer whale Ig, cells were ®rst incubated with the polyclonal anti-killer whale Ig (Bernadette Taylor, University of California, Davis, CA), and then incubated with FITC-conjugated F(ab0 )2 goat anti-rabbit IgG …H ‡ L† (Caltag, South San Francisco, CA). Subsequently, cells were incubated with mouse anti-cetacean, and labeled with PE-conjugated goat anti-MS IgG …H ‡ L† (Molecular Probe, Eugene, OR). Incubations were performed at 4 8C in the dark for 30 min. Cells were analyzed on a FACScan ¯ow cytometer (Becton Dickinson, Mountain View, CA). A gate for lymphocytes was determined according to relative size (forward scatter) and complexity (side scatter), and 5±10 000 electronically gated lymphocytes were collected and analyzed using markers adjusted according to proper controls. 2.6. Immunohistochemistry Diluted hybridoma culture supernatants were applied to frozen sections of common dolphin (D. delphis) lymph nodes. Antibody binding was detected using biotinylated horse anti-Ms IgG (Vector Laboratories, Burlingame, CA) followed by streptavidin-horseradish peroxidase (Zymed Laboratories, South San Francisco, CA) and DAB chromogen with H2O2 as substrate as previously described (Blanchard-Channell et al., 1994). 3. Results 3.1. Monoclonal antibodies Four mAbs (F21.B, F21.C, F21.F and F21.I) against bottlenose dolphin peripheral blood mononuclear cell surface proteins were produced and characterized. These antibodies were all IgG1 except for F21.B which was an IgG2a. 3.2. Immunoprecipitation of target antigen Immunoprecipitations were performed to determine the molecular weight of the antigen recognized by three out of four of our mAb (Fig. 1). F21.B precipitated proteins of 83, 112 and 127 kDa, F21.F precipitated a single protein of 144 kDa, and F21.I precipitated proteins of 43 and 59 kDa.

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Fig. 1. Immunoprecipitation of F21.I using common dolphin thymocytes, and F21.B and F21.F using common dolphin mesenteric lymph node lymphocytes. Each experiment included an irrelevant control antibody (left lane). Molecular weight (kDa) of specific bands is indicated.

3.3. Immunohistology Sections of cetacean lymph node were stained with the different mAb to con®rm their speci®city for B or T cell regions (Fig. 2). F21.B and F21.F consistently stained lymphocytes predominantly in follicles and rarely in the paracortex (Fig. 2A and B).

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Fig. 2. Sections of common dolphin lymph node stained with F21.I (A), F21.B (B) and F21.F (C) using immunoperoxidase. Sections were also stained with an irrelevant control mAb, which did not result in significant labeling (not shown).

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Table 1 Relative percentages of PBMC from different species of cetaceans labeled with the different mAbs Species

Killler whale (Orcinus orca) Bottlenose dolphin (T. truncatus) Beluga (Delphinapterus leucas) Striped dolphin (Stenella caeruleoalba) Pacific white-sided dolphin (Lagenorhinchus obliquidens) False killer whale (Pseudorca crassidens) Pilot whale (Globicephala melaena) Bridled dolphin (Stenella frontalis) Dall's porpoise (Phocoenoides dalli) Common dolphin (D. delphis)

Mean, standard deviation, No. of samples (n) %F21.B

%F21.C

%F21.F

%F21.I

24.47, 10.97, 290 14.11, 11.06, 22 16.86, 34.01, 15 10.6, 5.09, 6

55.52, 44.78, 73.51, 74.64,

26.07, 12.00, 290 22.63, 16.27, 22 1.87, 2.70, 15 10.2, 3.55, 6

54.91, 46.67, 71.53, 72.23,

5.25, 4.22, 5

76.93, 8.13, 5

14.22, 10.12, 5

71.89, 12.06, 5

4.21, 1.32, 2

90.94, 1.52, 2

4.81, 2.30, 2

91.9, 0.11, 2

5.80, 0.71, 2 9.20, ±, 1 19.30, ±, 1 25.60, ±, 1

59.10, 85.30, 70.00, 71.50,

4.55, 0.64, 2 13.70, ±, 1 18.50, ±, 1 30.60, ±, 1

56.90, 86.10, 68.30, 84.64,

16.43, 290 19.95, 9 8.75, 15 3.45, 6

27.29, 2 ±, 1 ±, 1 ±, 1

16.87, 289 17.95, 9 7.25, 14 10.13, 6

26.30, 2 ±, 1 ±, 1 ±, 1

Fig. 3. Representative histogram staining profiles of each mAb on single cell suspensions of common dolphin thymus and lymph node as well as killer whale PBMC. The bar represents the position of the marker to determine the percentage of cells that were labeled by each mAb.

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F21.I stained lymphocytes predominantly in the paracortex and rarely in follicles (Fig. 2C). Staining with the control mAb did not result in signi®cant labeling (not shown). 3.4. Flow cytometric analysis Relative percentages of PBMC from different species of cetaceans labeled with F21.B, F21.C, F21.F and F21.I are shown in Table 1. The mAbs against T-cell markers, F21.C and F21.I, cross-reacted with all the species tested. Mean values for F21.C and F21.I were consistent with each other within 2.5% for all species tested, except the Paci®c white-sided dolphin (5%). The T-cell marker mAbs reacted with 45±75% of PBMC in all the species in which they cross-reacted except for the bridled dolphin (85%) and the false killer whale (91%). The two B-cell marker mAbs, F21.B and F21.F, appeared to cross-react with all the species tested except for F21.F in beluga. Both the mAbs labeled a relatively similar proportion (2%) of PBMC in killer whale, striped dolphin, false killer whale, pilot whale and Dall's porpoise. F21.F labeled a higher proportion of PBMC than F21.B in bottlenose dolphin, Paci®c white-sided dolphin, bridled dolphin and common dolphin. F21.B but not F21.F-labeled PBMC in beluga whales. The B-cell marker mAbs labeled 4±31% of PBMC (except for F21.F in beluga). In the species where less than 10% of cells were stained with B cell marker mAbs, the percentage of positive cells stained with an irrelevant control mAb

Fig. 4. Double labeling of bottlenose dolphin splenocytes using selected pairs of mAbs. Cells were labeled with: (A) F21.F and goat anti-mouse Ig-PE and then F21.I-FITC; (B) F21.B and goat anti-mouse Ig-PE and then F21.I-FITC; (C) F21.B and goat anti-mouse Ig-PE and then F21.F-FITC; (D) F21.C and goat anti-mouse Ig-PE and then F21.I-FITC. Quadrants were adjusted according to proper controls.

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did not exceed 1% except for one striped dolphin (1.22%) and two Paci®c white-sided dolphins (1.14 and 3.00%). In all species studied, the sum of maximal reactivity of PBMC with a T-cell and a B-cell marker mAb never exceeded 100%. Representative histogram staining pro®les of each mAb on single cell suspensions of common dolphin thymus and lymph node as well as killer whale PBMC are shown in Fig. 3. F21.B and F21.F did not stain thymocytes, but did bind on approximately 63 and 65% of mesenteric lymph node cells, respectively. F21.C and F21.I both stained 100% of the thymocytes and approximately 46% of mesenteric lymph node cells. Blood PBMC staining percentages are detailed in Table 1. Double labeling of bottlenose dolphin splenocytes was performed to de®nitely assess the speci®c reactivity of our mAbs (Fig. 4). To study the relationship between B and T cell marker mAbs, cells were labeled with F21.I and either F21.F (Fig. 4A) or F21.B (Fig. 4B). While the proportion of double positive cells for F21.I and F21.B (2%; Fig. 4B) probably represented only background, that of double positive cells for F21.I and F21.F (5%; Fig. 4A) appeared distinct and signi®cant. A population of bottlenose dolphin splenocytes (18%) were negative for both F21.I and F21.B (Fig. 4B), while those double negative cells were scarce (4%) on double labeling with F21.I and F21.F (Fig. 4A).

Fig. 5. Double labeling of killer whale splenocytes using (A) F21.I or F21.F and goat anti-mouse Ig-PE and (B) a polyclonal antibody against killer whale immunoglobulins and goat anti-rabbit Ig-FITC.

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The relationship between the two different B-cell marker mAbs was investigated by labeling bottlenose dolphin splenocytes with F21.B and F21.F (Fig. 4C). 51% of the cells were labeled with both markers, while 13% were labeled by F21.F and not F21.B, and 34% were not labeled by any of the antibodies. Double labeling with the two T cell marker mAbs, F21.I and F21.C, showed that F21.C selectively inhibited the binding of F21.I (Fig. 4D), as F21.I would inhibit its own binding (data not shown), demonstrating speci®city and saturating concentrations of mAb. Inhibition of binding with any other combination of our mAbs was not observed (data not shown). In order to provide further evidence of the speci®city of the mAbs used, double labeling was performed on killer whale PBMC with F21.I or F21.F and anti-killer whale Ig (Fig. 5). While there were only 3% of the cells that expressed both F21.I and surface immunoglobulins, compared to 24% expressing surface immunoglobulins but not F21.I (Fig. 5A), 21% of the cells expressed both F21.F and surface immunoglobulins (Fig. 5B). In other words, most (78% or 21=…21 ‡ 6†) of the cells expressing F21.F simultaneously expressed surface immunoglobulins, and most (91% or 21=…21 ‡ 2†) of the cells expressing surface immunoglobulins simultaneously expressed F21.F. 4. Discussion The mAbs in this study recognize different subsets of peripheral blood lymphocytes. F21.B and F21.F did not react with thymocytes and stained follicles on lymph node sections; they were then assumed to recognize B lymphocytes. F21.C and F21.I reacted positively with 100% of thymocytes and stained lymphocytes in paracortical regions of lymph node sections; they were therefore assumed to recognize T lymphocytes. The speci®city of our mAbs for B and T cells is further supported by the fact that the B-cell mAbs and the T-cell mAbs were practically mutually exclusive on bottlenose dolphin spleen (Fig. 4B and C), that B cells (F21.F) but not T cells (F21.I) were positive for surface Ig (Fig. 5), and that the total of the maximum reactivity of PBMCs with a T-cell and B-cell mAb never exceeded 100%. In order to con®rm the speci®city of our mAbs, the molecular weight of the surface proteins they immunoprecipitated was determined. F21.B precipitated a protein of 83 kDa which is in the range of CD19 in humans (95 kDa) and mice (95 kDa), in which species CD19 is expressed on B cells from their early stage of development until plasma-cell differentiation (Tedder et al., 1994). The 127 kDa protein that was co-precipitated by F21.B could represent the whole CD19/CD81/Leu-13 complex …83 ‡ 26 ‡ 16 ˆ 125 kDa† (Tedder et al., 1994) which would not be completely reduced, or could correspond to the p130 which was co-precipitated with CD19 in other studies (Matsumoto et al., 1991; Bradbury et al., 1992, 1993). The 112 kDa protein co-precipitated could represent the CD19/CD81 complex …83 ‡ 26 ˆ 109 kDa†. The absence of CD81 (26 kDa) and Leu-13 (16 kDa) on the immunoprecipitation with anti-CD19 might be due to the reduced sensitivity of the technique used for low molecular weight proteins (Erickson et al., 1982) or might depend on the detergent used for membrane solubilization of the cells, which affected the stability of the complex and thereafter the co-precipitation results

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in other studies (Matsumoto et al., 1991; Bradbury et al., 1992). The chances of coprecipitating CD19 with a mAb to another protein of the complex such as CD81 or Leu-13 are small since the co-precipitations seem to be directional, i.e. anti-CD19 and anti-CD21 co-precipitate CD81 and Leu-13, but anti-CD81 and anti-Leu-13 do not co-precipitate CD19 and CD21 (Bradbury et al., 1992, 1993). This suggests F21.B may recognize CD19 and not CD81 or Leu-13. F21.F immunoprecipitated a single protein of 144 kDa. This is consistent with the 145 kDa protein immunoprecipitated with anti-CD21 in human, which is expressed on mature B cells (Tedder et al., 1994). No other protein of the CD19/CD21/CD81/Leu-13 complex (Tedder et al., 1994) was co-precipitated with F21.F. Once again, this might depend on the detergent used for membrane solubilization of the cells, which affected the stability of the complex and thereafter the co-precipitation results in other studies (Matsumoto et al., 1991; Bradbury et al., 1992). F21.I immunoprecipitated proteins of 43 and 59 kDa. This is in the range of the proteins precipitated by anti-CD2 in human (50±58 kDa; Brown et al., 1987), mice (55±65 kDa; Altevogt et al., 1989), pig (50 kDa; SaalmuÈller et al., 1994), bovine (48 and 58±62 kDa; Howard and Naessens, 1993), sheep (50±55 kDa; Mackay et al., 1988; 50 kDa; Hopkins et al., 1993) and horse (52 kDa; Tumas et al., 1994). The variability of the molecular weight of the protein immunoprecipitated with anti-CD2 can be explained by the fact that CD2 is made of a single polypeptide chain of molecular weight about 40 kDa which is extensively and variably N-glycosylated (Brown et al., 1987). Flow cytometric analysis also supports the hypothesis that F21.I may recognize a CD2 homologue. CD2 is one of the ®rst antigens expressed following colonization of the thymus by T-cell precursors and is detectable on the cell surface prior to the TCR/CD3 complex (Strominger, 1989). The unimodal staining of 100% thymocytes with CD2 was similar to what has been observed in human (Turka et al., 1992). Despite the high background in indirect staining with F21.I (Figs. 3 and 4), labeling with FITC-conjugated antibodies demonstrated that its antigen (putative CD2) was expressed only on T lymphocytes. This ®nding is in accordance with ®ndings in human (Moingeon et al., 1989), rats (Beyers et al., 1989) and horses (Tumas et al., 1994). Our results differ from a previous report in which an anti-bovine CD2 cross-reacted with nearly 100% of beluga lymphocytes (De Guise et al., 1997), as is the case in mice where CD2 is expressed at similar density on B and T cells (Altevogt et al., 1989). Some mAbs were characterized on the basis of their staining and inhibition of staining for mAbs that recognized the same epitope (Ling et al., 1987). One of our mAbs (F21.C) was not analyzed by immunoprecipitation but double labeling demonstrated its potential to selectively inhibit the binding of F21.I (Fig. 4D). These data, along with the very similar staining pattern of F21.C and F21.I on PBMC from different species and on single cell suspensions of lymph node and thymus, strongly suggest that F21.C may react with a similar or closely related epitope of CD2 as F21.I does. The last hypothesis would be favored by the fact that F21.I, but not F21.C, cross-reacted with murine splenocytes and peripheral blood lymphocyte (data not shown). F21.F (our putative anti-CD21 mAb) labeled a slightly higher percentage of cells than F21.B (our putative anti-CD19 mAb) on killer whale PBMC (Table 1), despite the fact that anti-CD21 should label only mature B lymphocytes while anti-CD19 should label B cells

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from their early stage of development until plasma-cell differentiation (Tedder et al., 1994). This could be explained by the fact that anti-CD21 labels not exclusively B cells but also some T lymphocytes (Fischer et al., 1991; Carter and Fearon, 1992). Double labeling con®rmed that 13% of bottlenose dolphin splenocytes were positive for F21.F and negative for F21.B, probably representing non-B cells (Fig. 4C). Furthermore, double labeling with F21.B and F21.I, a T-cell marker mAb, showed a population of bottlenose dolphin splenocytes (18%) that were negative for both F21.I and F21.B, representing non-T, non-B cells (Fig. 4B) while those double negative cells were scarce (4%) on double labeling with F21.I and F21.F (Fig. 4A). While the proportion of double positive cells for F21.I and F21.B (2%; Fig. 4B) probably represented only background, that of double positive cells for F21.I and F21.F (5%; Fig. 4A) seemed more signi®cant and probably represented the previously discussed non-B cells. Those data indicate that putative CD21 is possibly expressed not only on a few T lymphocytes but also on some non-B and non-T lymphocytes in the spleen of bottlenose dolphins. The wide cross-reactivity of our mAbs between different species of cetaceans is not surprising since they all belong to the same family (Delphinidae), except for beluga whale (Monodontidae) and Dall's porpoise (Phocoenidae), and considering the close genetic relationship within the Delphinidae (Milinkovitch et al., 1993). There are potential diagnostic and therapeutic uses associated with our mAbs. Chimeric anti-CD19 antibodies have been shown to have anti-tumor activity in vitro and in vivo (Pietersz et al., 1995). Anti-CD21 was shown to regulate IgE production (Aubry et al., 1992). These mAb should provide a understanding of the immune system of normal and diseased cetaceans. Acknowledgements The authors would like to thank the veterinarians of Sea World Parks (James McBain, Tom Reidarson, Leslie Dalton, Mike Walsh, Sam Dover) for providing blood samples from different species of cetaceans as well as San Pedro Marine Mammal Center, Randy Wells (Mote Marine Laboratory) and Sam Ridgway (NOSC) for helping in collection of cetacean tissues. SD is supported by a fellowship from the Medical Research Council of Canada. References Altevogt, P., Michaelis, M., Kyewski, B., 1989. Identical forms of the CD2 antigen expressed by mouse T and B lymphocytes. Eur. J. Immunol. 19, 1509±1512. Aubry, J.-P., Pochon, S., Graber, P., Jansen, K.U., Bonnefoy, J.-Y., 1992. CD21 is a ligand for CD23 and regulates IgE production. Nature 358, 505±507. Beyers, A.D., Barclay, A.N., Law, D.A., He, Q., Williams, A.F., 1989. Activation of T lymphocytes via monoclonal antibodies against rat cell surface antigens with particular reference to CD2 antigen. Immunol. Rev. 111, 59±77. Blanchard-Channell, M., Moore, P.F., Stott, J.L., 1994. Characterization of monoclonal antibodies specific for equine homologues of CD3 and CD5. Immunology 82, 548±554. Bradbury, L.E., Kansas, G.S., Levy, S., Evans, R.L., Tedder, T.F., 1992. The CD19/CD21 signal complex of human B lymphocytes includes the target of antiproliferative antibody-1 and Leu-13 molecules. J. Immunol. 149, 2841±2850.

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