Identification of canine T-lymphocyte subsets with monoclonal antibodies

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

187

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.

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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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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

4.78

1.140

12.125

8.53

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53_+ 13 <2 6+_ 4 5_+ 1 16_+ 5

74+7 13 12+4 26_+8 39_+8

70+11 6 12_+ 4 25_+ 8 34_+ 8

'The data represents the mean + SD of the percentage of positive lymphocytes in each tissue. Percent positive staining in the negative control was less than 2% in all samples.

eral blood, lymph node, spleen and thymus cells (Table 1 ). Monoclonal antibodies 1.140 and 4.78 reacted with a minor fraction of all lymphocytes tested and showed no detectable reactivity with peripheral blood granulocytes. The dislributions of reactivity as indicated by the FCM fluorescence histograms were similar for both monoclonals. Cross-blocking studies were performed where peripheral blood lymphocytes were stained with 1.140, 4.78 and 1.140 mixed with 4.78. The percentage of cells staining with the individual antibodies was within 5% of those stained with the mixture. In addition, the fluorescence intensity of the mixed antibody sample was not different from the intensity of the samples stained with the individual antibodies. To ensure that the fluorescence intensity data accurately reflected the m a x i m u m level of antibody binding, these tests were performed with saturating concentrations of monoclonal antibody and goat anti-mouse (heavy- and light-chain )-FITC. In a different series of tests, the binding of 1.140 blocked subsequent binding of FITC-conjugated 4.78. Monoclonal antibodies 8.53 and 12.125 reacted with a larger fraction of lymphocytes and with varying amounts of a minor fraction of granulocytes. The reactivity with granulocytes appears to be restricted to monocytes and neutrophils and was observed in 20% of the dogs evaluated. The percentage of cells stained with 8.53 or 12.125 was within 6% of each other stained separately or together, and the binding of 12.125 blocked the binding of 8.53-FITC. Monoclonal antibody 8.358 reacted with the largest fraction of lymphocytes tested with no detectable reactivity with granulocytes or bone marrow. The staining pattern of 8.358 was uniform with lymphocytes in the secondary lymphoid tissues tested, but variable in the thymus. Additive staining experiments were performed to further evaluate the relationship between the peripheral blood lymphocytes recognized by monoclonals 1.140/4.78, 8.53/12.125 and 8.358. The percentage of cells reacting with a mixture of 1.140 and 12.125 was within 5% of the sum of the percentage of cells that reacted separately with 1.140 and 12.125. The percentage of

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Fig. 2. Two-color analysis of canine peripheral blood reacted with (A) 8.358-FITC (green fluorescence) and goat anti-clog IgG heavy- and light-chain-Biotin-SAPE (red fluorescence), (B) 8.358-Bio-SAPE and 12.125-FITC, (C) 8.358-biotin-SAPE and 4.78-FITC, (D) 4.78-BiotinSAPE and 12.125-FITC.

cells reacting with 1.140 and 8.358 was similar to the percentage of cells reacted with only 8.358. This same relationship was observed when 12.125 and 8.358 were similarly tested. These data suggested that the populations recognized by 1.140/4.78 and 8.53/12.125 were exclusive of each other, and that they were inclusive of the 8.358-positive population. Two-color FCM analysis was performed to confirm the phenotypic rela-

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TABLE 2 Distribution of lymphocyte subsets in the peripheral blood of normal adult dogs

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tionship between populations recognized by 1.140, 4.78, 8.53, 12.125, 8.358 and goat anti-dog IgG heavy- and light-chain. In lymph node suspensions, all monoclonals reacted with cells that did not express surface immunoglobulin (data not shown). The relationship between the populations recognized by 4.78, 12.125 and 8.358 was then evaluated using peripheral blood lymphocytes (Fig. 2 ). Monoclonal 8.358 reacted with approximately 80% surface Ig negative lymphocytes (Fig. 2A ). More than 89% of the 12.125 positive cells also expressed the 8.358 antigen (Fig. 2B). The population recognized by 4.78 was inclusive of those recognized by 8.358 (Fig. 2C). Monoclonals 4.78 and 12.125 reacted with mutually exclusive populations (Fig. 2D ). Two-color analysis in the thymus showed that approximately 47% of cells co-expressed 4.78 and 12.125 (Fig. 3).

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Distribution of lymphocyte subsets in peripheral blood The frequency distribution of peripheral blood lymphocyte populations defined by 4.78, 12.125 and 8.358 was determined in a group of twenty-nine normal adult beagles. The proportions of cells defined by these monoclonals is similar to those values reported for pan-T-, CD4- and CD8-positive lymphocytes reported for the human and non-primate species (Table 2).

SDS-PAGE analysis of cell surface antigens Molecular weight of the antigens recognized by the mAb was determined by SDS-PAGE analysis of immunoprecipitated surface-labeled cell lysates. Under reducing conditions, the samples immunoprecipitated by 4.78 and 1.14 ran as a heterodimer of 32 kDa and 35 kDa peptides (Fig. 4, lanes 2 and 3 ). In three separate experiments using thymus cells, the 32 kDa band appeared to be very weak. In one experiment, where cells from a 1.140 +, 12.125-can-

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ine leukaemia were used as antigen source, the 35 kDa band was very strong, and the 32 kDa band was not evident (data not shown). The immunoprecipitate from 12.125 ran as a 56 kDa (Fig. 4, lane 4). Under non-reducing conditions, the immunoprecipitates from 1.140 and 4.78 showed a very weak band at around 70 kDa, with very strong bands occurring at ranges greater than 120 kDa. In the same experiments, the immunoprecipitate from 12.125 ran as a 56 kDa band (data not shown). There was no detectable band for the 8.53 lane under reduced or non-reduced conditions. DISCUSSION

Herein, we report the production and characterization of five mAb specific for canine T-lymphocytes. Monoclonal antibody 8.358 reacts with most or all T-lymphocytes in all tissues tested, 1.140 and 4.78 react with a minor subset of 8.358 positive cells, whereas 8.53 and 12.125 react with a major subset of the same population. The patterns of expression of the antigens recognized by 8.53/12.125 and 1.14/4.78, and the molecular weight analysis of the determinants defined by these reagents lead us to propose that these reagents react with canine equivalents to human CD4 and CD8, respectively. The stages of ontogeny of functional subsets of T-lymphocytes is manifested by the expression of cell surface markers that confer function and phenotype. The delineation of maturational stages within the thymus by cell surface phenotype has been carefully described in the mouse and human (Scollay et al., 1982; Lanier et al., 1986). The fundamental pattern of CD4 and CD8 expression in the thymus is consistent among all species where these molecules have been defined. CD4 and CD8 are co-expressed in more than 40% of thymocytes in the cat and 70% in the human, while in the peripheral blood, lymph node and spleen, only 2% or less of the cells co-express both antigens (Tompkins et al., 1990). We were able to demonstrate with one- and two-color FCM analysis that 1.140/4.78 and 8.53/12.125 are co-expressed in 47% of thymus cells and less than 1% of all secondary lymphoid tissues tested. In addition, the lymphocyte subsets defined by 1.140/4.78 and 8.53 / 12.125 were shown to be inclusive of those cells defined by 8.358. We provide evidence that the CD4-1ike molecule defined by 8.53 and 12.125 is not uniquely expressed on leukocytes of thymic origin. The FACS data demonstrate that monoclonals 8.53 and 12.125 have the same pattern of reactivity with blood granulocytes. These data are in support of the identification of the 8.53/12.128 molecule as being the canine homolog of h u m a n CD4 in that the h u m a n CD4 molecule has been identified on monocytes, brain tissue and eosinophils, and murine CD4 has been identified on monomyeloid precursors in bone marrow ( M a d d o n et al., 1986; Fredrickson and Basch, 1989; Riedel et al., 1990). In addition, CD4 m R N A has been isolated from murine B-lymphocytes, macrophages and brain tissues (Maddon et al., 1987 ). The proportional relationship between CD4- and CD8-positive lympho-

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cytes is similar in all species tested. Variations of the CD4:CD8 ratio in human and cat has been used as an index for immunomodulation or pathology associated with a variety of virus infections (Fahey et al., 1984; Novotney et al., 1990). The 12.125:4.78 ratio was determined to be 2.14 in twenty-nine healthy adult beagles. To confirm that mAb 1.140/4.78 and 8.53/12.125 recognize CD8- and CD4-1ike molecules, the reduced and non-reduced molecular weight of immunoprecipitates was determined. It was found that 1.140 and 4.78 precipitated a heterodimer that migrated as 32 kDa and 35 kDa peptides under reduced conditions. The intensity of the 32 kDa band was very weak in three different experiments using thymus cells as antigen. When a 1.140/4.78-positive canine leukaemia cell was used, the 32 kDa band was not evident, whereas the 35 kDa band was quite strong. In three experiments, the non-reduced bands were not visible at 70 kDa. However, there were several high molecular weight bands above 120 kDa. Because these high molecular weight bands were difficult to resolve from background, the evidence for a dimeric or multimeric form of the 1.140/4.78 antigen was not confirmed in four experiments. These data suggest that 1.140 and 4.78 react with 35 kDa peptide, though sequential immunoprecipitation studies would be necessary to confirm this. A 56 kDa band was precipitated by 12.125 under reduced and non-reduced conditions, whereas in three experiments, we were unable to precipitate any antigen with 8.53. This was not expected, since the binding of 12.125 blocked the binding of 8.53-FITC. In four immunoprecipitation experiments, there was no evidence that 8.358 precipitated labeled antigen. These data provide evidence for the delineation of canine T-lymphocytes as defined by monoclonal 8.358. Monoclonal 8.53 / 12.125 and 1.140/4.78 define cell-surface determinants that are structurally related to human CD4 and CD8, respectively. In addition, the tissue distribution of these antigens is remarkably consistent with the distribution of the functional and phenotypic subsets defined by CD4- and CD8like molecules in several mammalian species. CONCLUSION

Five monoclonal antibodies (mAb), specific for canine T-lymphocyte and T-lymphocyte subsets, were produced from fusions performed with mice immunized with canine lymph node and thymus ceils. One- and two-color flow cytometry of lymphoid tissues and SDS-PAGE analyses of immunoprecipitared ~25I surface-labeled thymus cell lysates were performed to evaluate the specificity of these mAb. Monoclonal antibody 8.358 reacted with surface immunoglobulin-negative lymphocytes in peripheral blood, spleen, and lymph node. Antibodies 1.140 and 4.78 reacted with approximately 18% peripheral blood lymphocytes, while antibodies 8.53 and 12.125 reacted with approximately 39% peripheral blood lymphocytes. The subsets defined by 1.140 and

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4.78 and the subsets defined by 8.53 and 12.125 were mutually exclusive and together accounted for almost all cells recognized by monoclonal antibody 8.358 in spleen, lymph node and peripheral blood. Approximately 47% thymus cells showed dual expression of 1.140/4.78 and 8.53 / 12.125. Antibodies 1.140 and 4.78 immunoprecipitated a 32-35 kDa heterodimer from radiolabeled thymus cell lysate under reducing conditions. Antibody 12.125 immunoprecipitated a single 56 kDa chain under reducing and non-reducing conditions. Thus, 8.53/12.125 and 1.140/4.78 react with populations of canine lymphocytes that are proportionally similar to cells expressing CD4- and CD8like molecules in several non-primate species. Furthermore, the molecules precipitated by 12.125 and 1.140/4.78 were similar in size and structure to human CD4 and CD8, respectively. The availability of mAb specific for canine T-cells and T-cell subsets expressing CD4 and CD8 homologs will enhance the study of the normal and pathological states of the canine immune system. ACKNOWLEDGMENTS

The authors wish to thank C. Hannaway, L. Schoemaker and K. Beegle for excellent technical assistance. The critical comments of the late Prof. Alan F. Williams are gratefully acknowledged; his influence on this field has been profound and will be sorely missed. REFERENCES Brideau, R.J., Carter, P.B., McMaster, W.R., Mason, D.W. and Williams, A.F., 1980. Two subsets of rat T lymphocytes defined with monoclonal antibodies. Eur. J. lmmunol., 10: 609615. Carter, P.B., Beegle, K.H. and Gebhard, D.H., 1986. Monoclonal antibodies: clinical uses and potential. Vet. Clin. North Am., 16:1171-1179. Chen, Y.X., Welte, K., Gebhard, D.H. and Evans, R.L., 1984. Induction of T-cell aggregation by antibody to a 16 kd human leukocyte differentiation antigen. J. lmmunol., 133: 24962501. Clark, S.J., Jefferies, W.A., Barclay, A.N., Gagnon, J. and Williams, A.F., 1987. Peptide and nucleotide sequences of rat CD4 (W3/25 ) antigen: Evidence for derivation from a structure with four immunoglobulin-related domains. Proc. Natl. Acad. Sci. USA, 84:1649-1653. Ellis, J.A., Baldwin, C.L., MacHugh, N.D., Bensaid, A., Teale, A.J., Goddeeris, B.M. and Morrison, W.I., 1986. Characterization by a monoclonal antibody and functional analysis of a subset of bovine T lymphocytes that express B6T8, a molecule analogous to human CD8. Immunology, 58: 351-358. Evans, R.L., Wall, D.W., Platsoucas, C.D., Siegal, F.P., Fikrig, S.M., Testa, C.M. and Good, R.A., 1981. Thymus-dependent membrane antigens in man: Inhibition of cell-mediated lympholysis by monoclonal antibodies to TH2 antigen. Proc. Natl. Acad. Sci. USA, 78: 544548. Fahey, J.L., Prince, H., Weaver, M., Groopman, J., Visscher, B., Schwartz, K. and Detels, R., 1984. Quantitative changes in T helper or T-suppressor/cytotoxic lymphocyte subsets that distinguish acquired immune deficiency syndrome from other immune subset disorders. Am. J. Med., 76: 95-100.

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Fredrickson, G.G. and Busch, R.S., 1989. L3T4 antigen expression by hemopoietic precursor cells. J. Exp. Med., 169: 1473-1478. Lanier, L.L., Allison, J.P. and Phillips, J.H., 1986. Correlation of cell surface antigen expression on human thymocytes by multi-color flow cytometric analysis: Implications for differentiation. J. Immunol., 137: 2501-2507. Ledbetter, J.A., Evans, R.L., Lipinski, M., Cunningham-Rundles, C., Good, R.A. and Herzenberg, L.A., 1981. Evolutionary conservation of surface molecules that distinguish T-lymphocyte helper/inducer and cytotoxic suppressor subpopulations in mouse and man. J. Exp. Med., 153: 310-323. Maddon, P.J., Dalgleish, A.G., McDougal, J.S., Clapham, P.R., Weiss, R.A. and Axel, R., 1986. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell, 47" 333-348. Maddon, P.J., Molineaux, S.M., Maddon, D.E., Zimmerman, K.A., Godfrey, M., Alt, F.W., Chess, L. and Axel, R., 1987. Structure and function of the human and mouse T4 genes. Proc. Natl. Acad. Sci. USA, 84: 9155-9159. Maddox, J.F., Mackay, C.R. and Brandon, M.R., 1985. Surface antigens, SBU-T4 and SBU-TS, of sheep T lymphocyte subsets defined by monoclonal antibodies. Immunology, 55: 739748. Nakauchi, H., Nolan, G.P., Hsu, C., Huang, H.S., Kavathas, P. and Herzenberg, L.A., 1985. Molecular cloning of Lyt-2, a membrane glycoprotein marking a subset of mouse T lymphocytes: Molecular homology to it human counterpart, Leu-2/TS, and to immunoglobulin variable regions. Proc. Natl. Acad. Sci. USA, 82:5126-5130. Novotney, C., English, R.V., Housman, J., Davidson, M.G., Nasisse, M.P., Jeng, C.R., Davis, W.C. and Tompkins, M.B., 1990. Lymphocyte population changes in cats naturally infected with feline immunodeficiency virus. AIDS, 4:1213-1218. Pescovitz, M.D., Lunney, J.K. and Sachs, D.H., 1984. Preparatiort and characterization of monoclonal antibodies reactive with porcine PBL. J. Immunol., 133" 368-376. Riedel, D., Lindemann, M., Brach, R., Mertelsmann, R. and Herrmann, F., 1990. Granulocytemacrophage colony-stimulating factor and interleukin-3 induce surface expression of interleukin-2 receptor p55-chain and CD4 by human eosinophils. Immunology, 70" 258-261. Scollay, R., Bartlett, P. and Shortman, K., 1982. T cell development in adult murine T-cell thymus: changes in expression of surface antigens Ly2, L3T4, and B2A2 during development from early precursor cells to emigrants. Immunol. Rev., 82: 79-85. Tompkins, M.B., Gebhard, D.H., Bingham, H.R., Hamilton, M.J., Davis, W.C. and Tompkins, W.A.F., 1990. Characterization of monoclonal antibodies to feline T lymphocytes and their use in the analysis of lymphocyte tissue distribution in the cat.'Vet. Immunol. Immunopathol., 26: 305-317.