Veterinary Immunology and Immunopathology, 33 (1992) 187-199

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Elsevier Science Publishers B.V., Amsterdam

Identification of canine T-lymphocyte subsets with monoclonal antibodies D.H. Gebhard and P.B. Carter Flow Cytometry/Hybridoma Facility, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA (Accepted 8 August 1991 )

ABSTRACT Gebhard, D.H. and Carter, P.B., 1992. Identification of canine T-lymphocyte subsets with monoclonal antibodies. Vet. Irnmunol. Immunopathol., 33:187-199. A panel of five murine monocional antibodies to canine T-lymphocytes were produced. Antibodies 4.78, 12.125 and 8.358 reacted with approximately 18%, 39% and 60% peripheral blood lymphocytes, respectively. Two color flow cytometric analysis showed that lymphocytes expressing 1.140, 4.78, 8.53 and 12.125 were subsets of lymphocytes expressing 8.358. The lymphocytes expressing 8.358 were negative for surface immunoglobulin.The subsets defined by 1.140, 4.78 or 8.53, 12.125 were mutually exclusive and together account for most ceNsexpressing 8.358 in the peripheral blood, spleen, and lymph node. In the thymus, approximately 47% cells were positive for both I. 140/4.78 and 8.53/ 12.125. SDS-PAGE analysis of radiolabelled thymus cell lysates demonstrated that antibodies 1.140 and 4.78 immunoprecipitated a 32,35 kd heterodimer under reducing conditions and 12.125 immunoprecipitated a single 56 kd chain under reducing and non-reducing conditions. Antibodies 8.53/ 12.125 and 1.140/4.78 react with canine lymphocyte populations that occur in proportions similar to lymphocytes expressing CD4 and CD8 like molecules in several primate and non-primate species. The molecules recognized by 12.125 and I. 140/4.78 were similar in size and subunit composition to human CD4 and CDS. ABBREVIATIONS BCS, bovine calf serum; FCM, flow cytometric; PBS, phosphate-buffered saline.

INTRODUCTION

The successful application of monoclonal antibody technology has led to the identification of functionally distinct subsets of morphologically similar lymphocyte cell populations in many mammalian species. Studies in the huCorrespondence to: Douglas H. Gebhard, Flow Cytometry/Hybridoma Facility, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00

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man, rat, mouse, pig, cow, cat and sheep suggest that molecules specific to functional subsets of lymphocytes are highly conserved during evolution (Brideau et al., 1980; Evans et al., 1981; Ledbetter et al., 1981; Pescovitz et al., 1984; Maddox et al., 1985; Ellis et al., 1986; Tompkins et al., 1990). In addition, investigators have demonstrated nucleotide sequence homology of CD8 in mouse and man, and CD4 in rat and man (Nakauchi et al., 1985; Clark et al., 1987). The identification of CD4 and CD8 homologs has occurred in several mammalian species, although an equivalent set of molecules has not been reported for the dog. Here we report the development and characterization of a panel of mAb that define T-lymphocytes and T-lymphocyte subsets expressing CD4- and CD8-1ike molecules in the dog. MATERIALS AND METHODS

Dogs Two groups of dogs were used as sources of peripheral blood, lymph node, spleen, thymus and bone marrow in this study. The first consisted of twentynine normal beagles bred and maintained at this facility. The second consisted of twenty-four puppies and adult dogs that were procured from an animal shelter after they had been slaughtered.

Separation of lymphocytes Peripheral blood leukocytes were isolated and enriched for the mononuclear fraction by discontinuous density gradient separation. Briefly, blood was collected via jugular puncture into heparin and diluted 1: 1 with phosphatebuffered saline (PBS). This was layered on Histopaque-1077 (Sigma Diagnostics, St. Louis, MO ) and centrifuged at 800 × g for 2 rain. The band of cells remaining at the interface was collected, washed three times in PBS containing 10% bovine calf serum (BCS) (Hyclone Laboratories, Logan, UT), and resuspended in RPMI- 1640 (GIBCO Laboratories, Grand Island, NY) with 5% BCS. The resulting fraction was more than 70% lymphocytes with viability greater than 90%. Bone marrow was recovered from the femur of canine cadavers and fractionated in the same way as the blood. Thymocytes, and lymphocytes from the spleen and lymph node, were isolated from solid tissue by teasing into RPMI-1640 with 5% BCS. The resulting cell suspension was separated on Histopaque-1077, washed, and maintained in RPMI-1640 with 5% BCS.

CANINE T-LYMPHOCYTE SUBSETS WITH MONOCLONAL ANTIBODIES

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Immunization and production of mAb Balb/c mize were immunized i.p. with a mixture of 4 × 107 isolated canine thymocytes and boosted every 2-3 weeks with a mixture of 107 thymus and lymph node cells. Three days after the fourth booster a mouse was killed, and its splenocytes were fused with the myeloma cell line P3 X 63-Ag8-653 (Carter et al., 1986). After 1-2 weeks growth, the supernatants were sampled, and flow cytometric (FCM) analysis was performed to identify those cultures which were producing antibody specific for subsets of an equal mixture of peripheral blood lymphocytes and thymus cells. The cultures that tested positive were expanded into larger cultures. The supernatants from the expanded wells were retested by FCM for reactivity with unmixed preparations of thymus cells and Ficoll (density = 1.119 )-separated peripheral blood leukocytes where specific analysis of lymphocytes or granulocytes could be made. The cultures that continued to produce antibodies specific for a distinct fraction of the lymphocyte populations tested were serially diluted at least 3 times to isolate a stable antibody-producing clone. The stable clones were expanded into larger volumes for production of high-titered supernatant and ascites fluid.

Isotype and subclass analysis of mAb An FCM-based assay was developed to determine the isotype and subclass of monoclonal antibody 1.140, 4.78, 8.53, 8.358 and 12.125. Briefly, 100 ~1 of exhausted antibody supernatant was reacted with 106 ( 100/zl) goat antimouse-conjugated beads (Simply Cellular Beads, Flow Cytometry Standards, RTP, NC) for 1 h at 4°C. The beads were washed in PBS twice at 150×g for 10 rain and then reacted for 1 h at 4 °C with fluorescein-conjugated goat antimouse specific for either IGM, IgG1, IgG2a, IgG2b or IgG3 (Southern Biotechnology, Birmingham, AL). The beads were washed twice, and FACS analysis was performed to measure the relative fluorescent intensity of the stained beads.

SDS-PAGE analysis of cell surface antigen was carried out Isolated canine thymocytes were surface iodinated by the lactoperoxidase method (Chen et al., 1984 ). Briefly, 1.5 × 108 viable cells were suspended in 1.5 ml PBS at 25 °C. To this, 400 #g lactoperoxidase (Sigma Chemicals, St. Louis, MO ) and 400/~Ci 125I (Amersham, Arlington Heights, IL) were added. While mixing, 50 #I 0.03% H202 was added three times, once every 5 min. The cells were then washed 3 times in cold PBS and solubilized for 30 min at 4°C in 3 ml lysis buffer pH 8.0 containing 1.0% w/v NP-40, 50 mM Tris, 150 mM NaC1, 5 mM EDTA, 25 mM PMSF, 0.02% NAN3. The suspension was

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then centrifuged at 800Xg for 5 min and passed through a 0.2 #m filter. The lysate was precleared by reacting it with 300 #1 packed Sepharose CL-4B beads coupled to protein A (Pharmacia LKB Biotechnology, Piscataway, N J) for 1 h at 4°C on a rotator. The immunoprecipitation was performed for 1 h at 4°C by reacting approximately 0.5 ml precleared lysate with 50/zl packed protein A Sepharose 4B beads previously coated with rat anti-mouse kappachain monoclonal antibody (HB 58, ATCC, Rockville, MD),then with mouse monoclonal antibody specific for the cell-surface antigen. The beads were washed 3 times in cold lysis buffer. The samples were eluted from the beads by boiling for 5 min in SDS-PAGE sample buffer and electrophoresed under reducing and non-reducing conditions in 10% SDS-PAGE gels. The gels were dried, and autoradiography was performed for 4 days with Kodak X-Omat film and intensifying screens.

Conjugation of mAb Purification and conjugation of mAb to either biotin or fluorescein were performed by Chromaprobe, Redwood City, CA.

Immunofluorescent analysis Lymphocyte cell surface analysis was performed on a FACScan equipped with Consort-30 software (Becton-Dickenson Immunocytometry Systems, Sunnyvale, CA). For indirect immunofluorescent analysis, 106 cells were incubated with each monoclonal antibody for at least 2 h at 4 ° C, washed twice in PBS, and then incubated with fluorescein (FITC)-conjugated goat antimouse IgG heavy- and light-chain-specific (Organon-Teknika, West Chester, PA) containing 20% heat-inactivated normal canine serum for 1 h at 4 °C. The cells were washed twice and resuspended in 0.5 ml PBS containing ethidium bromide and analyzed on the FACScan. For each sample, 4 parameter list mode data for 15 000 cells were collected, and gating was performed on forward angle scatter, side scatter, and red fluoresence (FL2) to allow the fluorescent analysis of viable lymphocytes. For two-color analysis, cells were reacted with FITC-conjugated monoclonals followed by the two-step incubation of biotin-conjugated monoclonals then streptavidin-phycoerythrin (SAPE) (Serotec, Kidlington, U K ) . Sample washing and preparation was performed as indicated above. List mode data were collected for 15 000 cells, and red and green fluorescent analysis was performed on lymphocytes gated by forward and side scatter. RESULTS

Production and characterization of mAb Twelve fusions were performed with spleens from mice immunized with canine lymph node and thymus cell suspensions. Flow cytometric analysis was performed to identify those cultures, secreting antibody specific for can-

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Fig. 1. FACS histograms of canine peripheral blood lymphocytes reacted with three monoclonals (solid line). (A) 8.358 (72% positive), (B) 12.125 (48% positive), (C) 4.78 (23% positive). The hatched line represents background staining. ine leukocyte subpopulations (Fig. 1 ). A FACS-adapted immunofluorescent assay was used to determine the isotype and subclass of each of the monoclonals. Monoclonal antibodies 4.78, 8.53 and 8.358 are of the IGM isotype; 1.140 and 12.125 are o f the IgG1 subclass. Monoclonals preferentially reactive with subsets o f lymphocytes were chosen for one- and two-color FCM analysis. FCM

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One-color FCM analysis was performed to determine the reactivity o f m A b 1.14, 4.78, 8.53 and 12.125 with bone marrow, lymphocytes from the periph-

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TABLE 1 Tissue distribution of lymphocyte subsets recognized by monoclonal antibodies Tissue

Thymus Bone marrow Spleen Lymphnode Peripheralblood

Percent positive antibody-staining lymphocytes 8.358

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12.125

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