Veterinary Immunology and Immunopathology, 27 ( 1991 ) 97-99 Elsevier Science Publishers B.V., Amsterdam

97

5.1 Workshop studies on the ovine CD 1 hgmologue

John Hopkins and Bernadette M. Dutia Department of Veterinary Pathology, University of Edinburgh, Edinburgh, UK

INTRODUCTION

CDI in humans consists of at least three separate polypeptides (Favaloro et al., 1987) which have been distinguished by sequential immunoprecipita- tion using monoclonal antibodies and by their slightly different tissue distri- butions (Cattoretti et al., 1987). All forms of human CD l are expressed on cortical thymocytes. In addition, CDla is expressed on epidermal/dermal Langerhans' cells (LC) and has a heavy chain with an apparent molecular weight (AMw) of 49 kDa. CD I b is expressed on dermal but not epidermal LC and has a 45 kDa AMw heavy chain. CD 1 c is expressed on epidermal and dermal LC and on peripheral blood B lymphocytes. It has a heavy chain with AMw 43 kDa. The differences in the molecular weight of the heavy chain between the various forms of the CD 1 molecule are due to differences in the extent of glycosylation (Calabi and Milstein, 1986). Monoclonal antibodies ~uu,,Lt~u to the workshop which had a known or putative reactivity to sheep CD l were compared.

MONOCLONAL ANTIBODIES

The monoclonal antibodies (and source laboratory) compared in these studies included CC13, CCI4 and CC20 (Compton); VPM5 (University of Edinburgh); TH97A (Pullman); 20-27 (University of Melbourne).

TISSUE DISTRIBUTION

The distribution of antigens recognised by mAbs CCI3, CCI4, CC20, TH97A and VPM5 appeared identical as assessed by immunohistology of sheep lymph nodes, spleen, thymus and skin and can be summarised as follows: - Lymph node, positive cells with a dendritic morphology were localised to

the paracortex. - Spleen, positive dentritic cells occurred in the periarteriolar region and in

the marginal zone.

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98 J. HOPKINS AND B.M. DUTIA

-Thymus, cortical thymocytes and dendritic cells in the medulla were positive. -Skin, positive dendritic cells occurred in the dermis and at the dermal/

epidermal junction. The mAb 20-27 reacted with the same cell populations as did the other five

antibodies but in addition it stained lymph nodes follicles and reacted with lymphocytes in the splenic marginal zone.

FACS ANALYSIS

FACS analysis of peripheral blood small lymphocytes (gated on the basis of ~'orward scatter [ FSC ] and side scatter [ SSC ] plots) and lymphocytes from afferent and efferent lymph resulted in almost identical data for CC 13, CC i 4, CC20, TH7A and VPM5 as follows: - PBL, 1-2% positive cells (up to 6% of high FSC cells). - Afferent lymph cells, 14-15% positive. These were cells with high FSC and high SSC. - Efferent lymph lymphocytes, < 1% positive.

The mAb 20-27 reacted with the high FSC/SSC cells in afferent lymph but also with 30-50% of small PBL, 4-5% of lymphocytes in afferent lymph and 20-25% oflymphocytes in efferent lymph.

IMMUNOPRECIPITATION

All the monoclonal antibodies immunoprecipitated two polypeptides which migrated with AMw of 44-46 kDa and 12 kDa where assessed by SDS-PAGE under reducing conditions. No difference was observed when non-reducing conditions were used (see Section 7 report by Dutia and Hopkins). Immu- nopurified antigen prepared using mAbs CC14 and VPM5 was used in an ELISA system to assess the cross-reactivity of the antibodies. All mAbs re- acted with both batches of purified antigen.

CONCLUSIONS

Monoclonal antibodies CC13, CC14, CC20, TH97A and VPM5 form a cluster of mAbs that, by analogy with the human data, recognise the sheep CDlb molecule. The mAb 20-27 did not cluster tightly with the other re- agents, due to its additional reactivity with sheep B cells (Mackay et a!., 1985 ). The cellular reactivity of mAb 20-27 suggests that it identifies the sheep hom- ologue of CDIc. However, this mAb clearly cross-reacts biochemically with the other reagents and therefore may be regarded as being pan-CD 1.

THE OVINE CDI HOMOLOGUE 99

REFERENCES

Calabi, F. and Milstein, C., 1986. A major family of human major histocompatibility complex- related genes not mapping to chromosome 6. Nature, 323:541-543.

Cattoretti, G., Berti, E., Mancuso, A., D'Amato, L., Shiro, R., Soligo, D. and Delia, D., 1987. An MHC Class I related family of antigens with widespread distribution on resting and ac- tivated cells. In: A.J. McMichael (Editor), Leucocyte Typing III, White Cell Differentiation Antigens. Oxford University Press, Oxford, pp. 89-91.

Favaloro, E.J., Grimsley, P., Kamath, S., George, V., Henniker, A. and Bradstock, K.F., 1987. Heterogeneity of antigens within CD I. In: A.J. McMichael (Editor), Leucocyte Typing III, White Cell Differentiation Antigens. Oxford University Press, Oxford, pp. 77-79.

Mackay, C.R., Maddox, J.F., ¢o..;golin-Ewens, K.J. and Brandon, M.R., 1985. Characterisation of two sheep lymphocyte differentiation antigens, SBU-T1 and SBU-T6. Immunology, 55: 729-737.