Veterinary Immunology and ImmunopathGlogy, 27 ( 1991 ) 101-102 Elsevier Science Publishers B.V., Amsterdam

101

5.2 Workshop studies on the ovine CD4 homologue

John Hopkins Department of Veterinary Pathology, University qf Edinburgh, Edinburgh, UK

INTRODUCTION

In humans and mice, the CD4 molecule is a single chain transmembrane glycoprotein of apparent molecular weight (AMw) 55 kDa (Maddon et al., 1985 ); the biological function of which seems to be an interaction with MHC class II (Doyle and Strominger, 1987; Gay et al., 1987). T cells expressing CD4 show restricted recognition of antigen in association with MHC class II (Marrack et al., 1983) and have mainly helper/inducer functions (Swain, 1983). A standardised comparison was made of the monoclonal antibodies submitted to the workshop which were considered to identify the sheep hom- ologue of CD4.

MONOCLONAL ANTIBODIES

The monoclonal antibodies used (and source laboratory) included ST4a (Basel Institute for Immunology; Mackay et al., 1988 ); GC l A l and GC50A l (Pullman); 44-38 and 44-97 (University of Melbourne; Maddox et al., 1985 ).

TISSUE DISTRIBUTION

The distribution of the antigens recognised by these five monoclonal anti- bodies was identical when assessed by immunohistology of sheep lymph nodes, spleen and thymus. The mAb GC I A l, submitted as an anti-bovine reagent, did not react on bovine tissues although it did stain sheep tissues~ The distri- bution of positive lymphocytes can be summarised as follows: - Lymph node, positive cells mainly in the interfollicular paracortex, few cells

within the follicles and small numbers in the medullary cords. - Spleen, positive cells localised in periarteriolar lymphoid sheath of the white pulp. - Thymus, nearly all cortical thymocytes and about 50% of the medullary

thymocytes appeared positive.

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102 J. HOPKINS

FACS ANALYSIS

FACS analysis of sheep PBL, lymphocytes from afferent and efferent lymph and thymocytes gave virtually identical results for all antibodies. Again, GCIA l did not react with bovine cells but gave a pattern of staining similar to the other mAbs when tested on sheep cells. The proportion of positive lym- phe, cytes detected in the various samples was as follows: - Peripheral blood lymphocytes, 25-40% - Afferent lymph, 49-57% - Efferent lymph, 54-56% - Thymus (newborn), 68-80% - Thymus (day 7), 44-54%

IMMUNOPRECIPITATION OF TARGET MOLECULES

Immunoprecipitation of target molecules from lymphocyte lysates was at- tempted with mAbs 44-38 and 44-97. Both mAbs immunoprecipitated a pro- tein which migrated as a single band with AMw 56 kDa when analysed by SDS-PAGE under reducing conditions.

CONCLUSIONS

The monoclonal antibodies ST4a, GC1A1, GC50AI, 44-38 and 44-97 clus- tered and, by analogy with human data, can be regarded as recognising the sheep homologue of CD4.

REFERENCES

Doyle, C. and Strominger, J.L., 1987. Interaction between CD4 and class II molecules mediates cell adhesion. Nature, 330: 256-259.

Gay, D., Maddon, P.J., Sekaly, R., Talle, M.A., Godfrey, M., Long, E., Goldstein, E., Chess, L., Axel, R., Kappler, J. and Marrack, P., 1987. Functional interaction between the human T cell protein CD4 and the major histocompatibility complex HLA-DR antigen. Nature, 328: 626-629.

Mackay, C.R., Hein, W.R., Brown, M.H. and Matzinger, P., 1988. Unusual expression of CD2 in sheep implications for T cell interactions. Eur. J. Immunol., 18:1681-1688.

Maddon, P.J., Littman, D.R., Godfrey, M., Maddon, D.E., Chess, L. and Axel, R., 1985. The isolation and nucleotide sequence ofa cDNA encoding the T cell protein T4: a new member of the immunoglobulin gene family. Cell., 42: 93-104.

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: 739- 748.

Marrack, P., Endres, R., Shimonkevitz, R., Zlotnik, A., Dialynas, D., Fitch, F. and Kappler, J., 1983. The major histocompatibility complex-restricted antigen receptor on T cells. II. Role of the L3T4 product. J. Exp. Med., 158: 1077-1091.

Swain, S.L., 1983. T cell subsets and recognition of MHC class. Immunol. Rev., 74: 129-142.