Monoclonal antibodies to a high molecular weight isoform of porcine CD45: biochemical and tissue distribution analyses

Veterinary immunology and immunopathology

Veterinary Immunology and lmmunopathology 56(1997) 151-162

ELSEVIER

Monoclonal antibodies to a high molecular weight isoform of porcine CD45: biochemical and tissue distribution analyses Rosario Bullido, Manuel G6mez de1 Moral, Nieves Domknech, Fernando Alonso, Angel Ezquerra, Javier Dominguez * Centro rlr Inuestt~ucititt

en Sunidd

Animul. IN/A, Valdeolmos 28130, Madrid.

Received I2 January 1996; accepted 2 I

Spuin

June 1996

Abstract

This report describes the obtention and characterization of two monoclonal antibodies (mAbs), 6E3/7 [mAb 6E3/7 was submitted to the Second International Swine CD Workshop, where it has been assigned to CD45R] and 3C3/9, which recognize the isoform of highest molecular weight of porcine CD45. This conclusion is based on their cell reactivity and tissue distribution, identical to that reported for the human high molecular weight isoform of CD45, and on data from immunoprecipitation and immunoblotting analyses which show that these mAbs react with the largest polypeptide of those precipitated by mAb 2A.5, that recognizes an epitope shared by all CD45 isofotms. These mAbs react with 60% of peripheral blood mononuclear cells (PBMC) but not with alveolar macrophages, granulocytes, platelets or erythrocytes. Antigen expression on PBMC is heterogeneous and is reduced after in vitro activation with mitogens. B cells and CDS+ T cells express more antigen than CD4+ T ceils. Using immunoperoxidase techniques, the antigen was detected on B cell areas of lymph nodes and Peyer’s patches, and on a subpopulation of medullary thymocytes. These mAbs will be useful reagents for functional and phenotypic analysis of porcine lymphoid cell populations by flow cytometry and immunohistochemistry. 0 1997 Elsevier Science B.V. Keywort/st

CD45R;

Abbreviations: interleukin;

Leukocyte

BSA,

bovine

mAb. monoclonal

1 Corresponding 0 1652427/97/$17.00

common

serum

antibody:

antigen;

Monoclonal

albumin; PBMC,

Con

peripheral

antlbodles:

A.

concanavalin

0

I997 El scvicr

Science B.V.

A;

blood mononuclear

author.

1’11 SOl65-2427(96)05728-S

Lymphocytes;

All rights reserved

FCS, cells

Pig

fetal

calf

serum;

IL,

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1. Introduction

CD45, also known as leukocyte common antigen, is an abundant cell surface glycoprotein expressed on all leukocytes. The molecule shows a marked structural heterogeneity with multiple isoforms (ranging from 180 to 220 kDa) resulting from the alternative splicing of three exons from a common precursor RNA that can generate at least eight different mRNAs. Cell-type specific glycosylation, which may modify both N- and O-linked carbohydrate structures, adds further diversity. Functionally, the molecule shows tyrosine phosphatase activity, and is required for normal antigen-induced lymphocyte activation (Trowbridge, 199 1; Trowbridge and Thomas, 1994). The different CD45 isoforms show a characteristic pattern of cellular distribution, which is conserved throughout mammalian evolution (Thomas, 1989). B cells express predominantly the largest isoform, thymocytes the smallest, and T cells different patterns of isoforms that appear to correlate with their function and prior exposure to antigen. Thus, naive T cells express high molecular weight isoforms, whereas memory T cells express the lowest molecular weight isoform (CD45RO) (Mackay, 1993). A panel of monoclonal antibodies (mAbs) to porcine CD45 molecules has recently been characterized by Zuckermann et al. (1994a,b). Four of these mAbs recognize specifically the isoforms of 210 and 226 kDa. In this paper we describe the generation and characterization of two mAbs which, according to biochemical, flow cytometric, immunohistochemical and functional analyses, recognize the highest molecular weight isoform of porcine CD45.

2. Materials

and methods

2.1. Animals and cells Large White pigs weighing between 30 and 40 kg were used as donors of blood and tissues. Peripheral blood mononuclear cells (PBMC) were isolated on Percoll discontinuous gradients after blood sedimentation in dextran as has been previously described (Gonzalez et al., 1990). Granulocytes were recovered from the lower Percoll phase. Residual erythrocytes were lysed by hypotonic treatment. Platelets were obtained from the platelet-rich plasma fraction resulting from the centrifugation of normal swine blood for 30 min at 350 x g. Alveolar macrophages were collected by bronchoalveolar lavage as described by Carrascosa et al. (1982) washed with Hanks buffer containing 2 mM 10% EDTA, resuspended at 5 X IO7 cells ml - ’ in fetal calf serum (FCS) containing dimethyl sulphoxide, and frozen in liquid nitrogen until use. Analyses of antigen expression during cell activation were performed on PBMC stimulated with 2.5 /-~g ml-’ concanavalin A (Con A; Sigma, USA) and cultured for 7 days. On days 3 and 6, cultures were fed with fresh medium (RPMI-10% FCS) containing 20% interleukin 2 (IL-2).conditioned medium. IL-2-conditioned medium was the supernatant of PBMC stimulated with 2.5 /*g ml ’ Con A for 2 days.

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PBMC from human, horse, cattle and dog were isolated by Ficoll-Hypaque gradient centrifugation.

2.2. Monoclonal

153

density

antibody production

The mAbs described in this study were derived from two fusions of X63-Ag.8.653 myeloma cells with spleen cells from Balb/c mice immunized with 2-day Con A blasts using standard procedures (Kohler and Milstein, 1975). Supematants were tested for activity against Con A blasts and resting PBMC by flow cytofluorometry (FACS). Cells from positive wells were cloned at least twice by limiting dilution. Class and subclass of mAbs were determined by ELISA, using rabbit antisera specific for mouse heavy and light chains and a peroxidase-conjugated goat anti-rabbit Ig (Bio-Rad, USA). Both mAbs were of IgGl, K isotype. For labellin g, mAbs were purified from ascitic fluid by affinity chromatography with Protein A-Sepharose CL 4B (Pharmacia, Sweden). Biotinylation of mAbs was performed as described previously (Dominguez et al,, 1990). mAb 2A5, isotype IgGl, was produced as described previously from the spleen of a mouse that had been inoculated with porcine lymphocytes. This mAb was analysed in the First International Swine CD Workshop and reacts with a common determinant of porcine CD45 (Zuckermann et al., 1994a). mAbs to porcine CD4 (74-l 2-4) and CDS (76-2-l 1) were kindly provided by Dr. J. Lunney CARS, USDA, Beltsville, USA). mAbs to porcine CD25 (K231-3B2) and light chains of porcine immunoglobulins (Kl39-3El) were kindly provided by Dr. C. Stokes (University of Bristol, UK).

2.3. Immunoprecipitation

analysis

PBMC (10’) were washed three times in phosphate buffered saline (PBS) and resuspended in 5 ml of PBS. Sulfo-NHS-biotin (Pierce, USA) (0.4 mg ml- ‘, final concentration) was added to the cells and incubated for 15 min at 4°C. After washing three times with PBS, cells were lysed with I ml of lysis buffer consisting in 10 mM Tris-HCI, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP40, 1 mg ml-’ bovine serum albumin (BSA), 0.2 U ml-’ aprotinin and 1 mM phenylmethylsulphonylfluoride. The lysate was precleared twice with 50 ~1 of a 25% (v/v> suspension of Protein G-Sepharose (Pharmacia, Sweden) in lysis buffer, and then incubated with the different mAbs. To 0.3 ml of hybridoma supernatant was added 0.1 ml of lysate sample and incubated for 2 h at room temperature. Then, 40 ~1 of a 25% (v/v> suspension of Protein G-Sepharose was added and incubated for 1 h with gentle mixing. Beads were washed three times with lysis buffer then boiled in electrophoresis sample buffer (0.062 M Tris-HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 0.001% bromophenol blue, with or without 0.7 M 2-mercaptoethanol); supernatants were run in SDS-7.5% PAGE (polyacrylamide gel electrophoresis) and transferred to nitrocellulose. Filters were incubated with streptavidin-peroxidase (Pierce, USA) and bands visualized by the ECL detection assay following the manufacturer’s indications (Amersham. UK).

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2.4. Immunoblotting PBMC (5 X IO71 were washed twice with PBS and solubilized in 0.5 ml of lysis buffer for 1 h at 4°C. After centrifugation at 12 000 X g for 30 min, the supematant was mixed with the sample buffer and run on SDS-7.5% PAGE under reducing conditions. Alternatively, PBMC lysates were subjected to immunoprecipitation with mAb 2A5, 6E3/7 or 3C3/9, and the bound proteins resolved by SDS-7.5% PAGE under reducing conditions. The separated proteins were transferred to nitrocellulose. Free binding sites on nitrocellulose were blocked with PBS-2% powdered milk. Thereafter, strips were incubated with hybridoma supematants, diluted at l/2 in PBS containing 0.2% Tween with 20 and 1% BSA, for 1 h at room temperature, followed by 1 h incubation peroxidase-labelled rabbit anti-mouse Ig (Dako, Denmark). Peroxidase activity was visualized using the ECL detection assay (Amersham, England). 2.5. Flow cytofluorometry Cells (5 X 105) were incubated on ice with 50 ~1 of hybridoma supematant for 30 min. After two washes in PBS containing 0.1% BSA and 0.1% sodium azide (fluorescence buffer, FB), cells were incubated for 30 min at 4°C with 50 ~1 of FITC-conjugated rabbit F(ab’), anti-mouse Ig (Dako, Denmark) diluted l/40 in FB. Cells were washed three times in FB and fixed in 0.3% paraformaldehyde prior to analysis in a FACScan (Becton Dickinson, USA). For two-colour analysis, PBMC were incubated with mAbs against porcine CD4, CD8 or Ig light chains, followed, after washin g, by rabbit F(ab’), anti-mouse Ig-FITC. Free binding sites were blocked with 5% normal mouse serum. Finally, cells were stained with biotin-labelled mAb 3C3/9 or 6E3/7 and streptavidin-phycoerythrin (Southern Biotechnology, USA). For the blocking studies, cells were incubated with 25 ~1 of hybridoma supernatants. After 10 min incubation at 4”C, and without washing, 25 ~1 of the optimal dilutions of biotin-labelled mAbs were added for an additional 30 min. Cells were then washed and stained with streptavidin-phycoerythrin for 10 min. An irrelevant, isotype-matched mAb was used as negative control. 2.6. Immunohistochemical

studies

Porcine tissues were snap-frozen in isopentane/liquid nitrogen and stored at -70°C. Frozen sections of 6 pm were fixed in cold acetone for 10 min and then washed with PBS. Sections were stained by an indirect immunoperoxidase technique as previously described (Minguez et al., 19881, and counterstained with methylene blue.

3. Results 3.1. Cellular and tissue distribution

of antigens recognized by mAbs 3C3/

9 and 6E3 / 7

The reactivity of mAbs 6E3/7 and 3C3/9 with PBMC and lymphoid organ cell suspensions was established by flow cytometry. Both mAbs showed similar staining

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3c3/9

6E3/7

PBLs

1

4g , 23,

28

,

Lymph nodes

Spleen

501

Thymus

Fluorescence Fig. I. Flow cytometric different within

lymphoid the histogram

analysis of the expression

tissues. A representative regions defined

of 6E3/7

expcrlment

by horizontal

intensity and 3C3/9

is shown.

antigens on PBMC

Numbers

indicate

and on cells from

the percentage

of cells

bars.

patterns (Fig. 1). About 60% of PBMC were positive, showing a bimodal distribution, with a bright and a dull population, comprising 17% and 43% of cells, respectively. AS noted in PBMC, two positive populations with different antigen density were also observed among lymph node and spleen cells. In contrast, only a single positive peak of low intensity, which represented approximately 18-26% of the population, was noted when thymocytes were stained with 3C3/9 or 6E3/7 mAbs. Alveolar macrophages, granulocytes, platelets and erythrocytes were negative (data not shown).

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6E3/7

CD8

CD4

L chains

control

Green fluorescence Fig. 3. Two-colour

flow cytometric analysis of 3C3/9

markers. PBMC were stained with anti-CD4 (74-12-41, irrelevant mAb (control) followed by FITC-labelled

or 6E3/7

expression versus other porcine lymphocyte

anti-CD8 (76-2.

rabbit anti-mouse

I I ), anti-light chains (Kl39-38 Ig, biotin-conjugated 3C3/9 or

I) or an 683/7.

and streptavidin- phycoerythrin. Numbers indicate the percentage of cells within the respective regions.

Tissue distribution of these antigens was also analysed by immunoperoxidase staining (Fig. 2). When frozen sections of thymus were stained with these mAbs, positive cells were located mainly in the medulla, although a few positive cells were also found scattered in the cortex. In lymph nodes, tonsil and Peyer’s patches, both mantle zone and germinal centres of follicles were strongly stained. In addition, a small number of cells scattered in the paracortical area were also stained. The distribution of the 6E3/7 and 3C3/9 antigens on PBMC was further studied by two-colour flow cytometry analysis using biotin-labelled mAbs 6E3/7 or 3C3/9 in combination with mAbs against porcine CD4, CD8 and Ig light chains (Fig. 3). The majority of B cells, defined by surface expression of Ig, as well as a subpopulation of CD8 T cells, were contained in the bright population of 6E3/7 or 3C3/9 positive cells. The CD8 dull population, which has previously been shown to contain the CD4-CD8 double positive cells (Pescovitz et al., 1985, 19941, and the CD4 population were for the most part restricted to 6E3/7 and 3C3/9 dull or negative cells. 3.2. Molecular

characterization

mAbs 3C3/9 and 6E3/7 precipitated a single polypeptide band of about 220 kDa from biotin-labelled PBMC lysates under both non-reducing (Fig. 4(A)) and reducing conditions (data not shown). This band comigrated with the largest polypeptide recognized by an anti-CD45 mAb (2A.5). A single band of about 220 kDa was also detected

Fig. 2. Cryostat sections of porcine thymus (A), lymph node (B) and Peyer’s patches (0 6E3/7

(A) and 3C3/9

(B and Cl by the immunoperoxidase

stained with mAbs

method. The section of the thymus shows the

cortical (cl and medullary (ml areas as indicated. In lymph node and Peyer’s patches the follicles (f) and interfollicular areas (if) are indicated. (A and C. X 40; B, X 100).

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204-

12182 -

B 1

2

3

4

5

-0

Fig. 4. (A) Analysis

by immunoprecipitation

were surface-labelled

with biotin,

described

2. Lane

Arrows

in Section indicate

subjected

the positions

I, irrelevant

mAb;

of the different

to immunoprecipitation

with

proteins were resolved by SDS-7.5% mAb 6E3/7

of the antigens recognized

lysed and analysed

(lanes 2 and 4) or 3C3/9

mAb

by SDS-7.5%

lane 2. 3C3/9;

components 2A5

(lanes

213

-

123

-

85

by mAbs 6E3/7

PAGE

3C3/9

complex. (lane

PAGE and analysed by immunoblotting (lanes 3 and 5). The position

and 3C3/9.

under non-reducing

lane 3, 6E3/7;

of the CD45 l-3),

_

as

lane 4, 2A5 (anti-CD45). (B) PBMC

4) or 6E3/7

lysates

were

(lane 5). Bound

with an irrelevant

of the molecular

PBMC

conditions

mAb (lane I),

weight markers are given

at the side of the figures.

by immunoblotting (not shown), indicatin g that the epitopes recognized by these mAbs remain intact after SDS denaturation. To further investigate the relationship between these bands, PBMC lysates were immunoprecipitated with mAb 2A5, 6E3/7 or 3C3/9, followed by immunoblotting with mAb 6E3/7 or 3C3/9. In all cases, the band of 220 kDa was detected, confirming the identity and CD45 specificity of the molecules recognized by mAbs 6E3/7 and 3C3/9 (Fig. 4(B)). 3.3. Epitope mapping Blocking experiments were performed for the characterization of the epitopes recognized by these mAbs. While 3C3/9 culture supernatant completely inhibited the binding

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A

Fluorescence intensity Fig. 5. Epitope analysis. Panel represents histograms from PBMC mAbs (I, irrelevant mAb; 2, 3C3/9; (B)

and avidin-phycoerythrin.

3, 6E3/7)

preincubated for IO min with unlabelled

followed by staining with 6E3/7-biotin

The tilled histogram corresponds to background

(A) or 3C3/9-biotin

staining with fluorescent

conjugate alone.

of biotin-labelled 3C3/9 to the cells, as determined by F.4CS analysis, 6E3/7 supernatant was unable to block it (Fig. 5). Similarly, 6E3/7 culture supematant, but not 3C3/9, inhibited the binding of bibtin-labelled 6E3/7 to the cells. These results indicate that these mAbs react with two different epitopes on the CD45 molecule. No inhibition was observed with mAb 2A5 on the binding of biotin-labelled 6E3/7 or 3C3/9 to the cells (data not shown), which indicates that this mAb binds to a different site on the CD45 molecule.

3.4. Analysis of antigen expression

during cell activation

PBMC were stimulated with Con A and cultured for 7 days. Changes in the surface phenotype of these cells were analysed by flow cytometry at different times. Activation of PBMC resulted in the expected expression of CD25 (Fig. 6) and increase in cell size (data not shown). A progressive decline was observed in the expression of 3C3/9 antigen (CD45R), which by day 7 was almost undetectable. In contrast, CD45 expression remained unmodified or slightly increased over this time.

160

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CD25

CD45

3c3t9

day 0

day 3

day 7

Fuorescence Fig. 6. Changes

in expression

period of 7 days, on PBMC

of 3C3/9

stimulated

immunofluorescence

using FITC-conjugated with fluorescent

3.5. Cross-reactions

on other

CD45

(mAb

with Con A (2.5 pug ml-

background

staining

(CD45R).

intensity

rabbit F(ab’),

conjugate

2A.5) and CD25

’). Surface

anti-mouse

(mAb

K231-3B2)

over a

antigens were detected by indirect

lg. The filled

histogram

corresponds

to

alone.

species

Reactivity of mAb 6E3/7 or 3C3/9 with leukocytes from different species, including human, horse, cattle and dog, was analysed by flow cytometry. None of these leukocytes was recognized by mAb 6E3/7 or 3C3/9 (data not shown).

4. Discussion This paper describes the characterization of two novel mAbs, named 3C3/9 and 6E3/7, raised against 2-day Con A-stimulated porcine lymphocytes. Based on biochemical, histochemical and flow cytometric analysis, the antigen recognized by these mAbs appears to be the analogue of the largest isoform of human CD45 (Pulido et al., 1988; Thomas, 1989). A clear demonstration of the CD45R specificity of these mAbs was their reactivity in immunoblotting with the largest component of the polypeptide complex immunoprecipitated by mAb 2A5, which recognizes an epitope shared by all CD45 isoforms. Isoforms of CD45 proteins that are expressed on a restricted group of cell types are designated CD45R. Because individual exons are used in more than one isoform, most mAbs specific for restricted CD45 epitopes usually recognize several isoforms. This probably occurs with mAbs 3C3/9 and 6E3/7, although only a single band was

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161

detected by immunoprecipitation and by immunoblotting. At least eight different CD45 isoforms can be generated through the alternative splicing of three extracellular exons; however, only a few bands can be resolved by SDS-PAGE. In man and mouse, four bands can be detected by SDS-PAGE, whereas in pig, as in sheep and cattle, only three can be found (Zuckermann et al., 1994b). Therefore, more than one isoform is likely to be present in some of these bands. In human, mouse and rat, the antibodies that recognize epitopes dependent upon products of the three alternative exons 4, 5 and 6 have been termed CD45RA, -B, and -C, respectively. CD45RO was defined by antibodies that specifically recognize the isoform derived by the removal of these three exons. According to the molecular weight and cell distribution of the antigen that they recognize, mAbs 3C3/9 and 6E3/7 seem to be of CD45RA specificity; however, a definitive assignation must await the availability of cell transfectants expressing individual isoforms of porcine CD45. In man, mouse and cattle, isoforms of CD45 have been used to distinguish different functional subsets of T cells (Sanders et al., 1988; Bottomly et al., 1989; Howard et al., 1991). Thus, the expression of CD45RA and CD45R0, respectively the high and low molecular weight isoforms of CD45R, has been correlated with their naive or memory status, although later studies suggest a closer link with the state of cell activation (Beverly, 1992). Evidence of bi-directional conversion between CD45RA and CD45RO phenotypes derives from in vivo and in vitro experiments (Bell and Sparshott, 1990; Warren and Skipsey, 1991; Michie et al., 1992). Cyclic expression of CD45RA has been observed during the culture of a CD45RO T cell line (Rothstein et al., 1991). Following the in vitro stimulation of T cells with a mitogen such as phytohaemagglutinin, the CD45 isoform expression changes, showin g a gradual decrease of CD45RA expression and a concomitant acquisition of the smallest isoforms (Serra et al., 1988; Deans et al., 1989; Wallace and Beverly, 1990). A similar phenomenon seems to occur in the pig, where expression of the 3C3/9 antigen gradually decreased after Con A stimulation of PBMC, being almost undetectable by day 7, while the expression of CD45 remained unaltered. Remarkably, these mAbs were derived from fusions where 2-day Con A-activated cells, which show a reduced expression of this antigen, were used as immunogen. In conclusion, we have produced and characterized two mAbs that recognize the largest isoform of porcine CD45. These mAbs can be useful in phenotypic and functional analyses of porcine lymphoid cell subpopulations.

Acknowledgements This work was supported by INIA Grant SC93- 155 and EC Project AIR3-CT93- 1332. R. Bullido and M. Gomez de1 Moral are recipients of INIA and CICYT Ph.D. fellowships, respectively.

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with