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S y m p o s i u m 6

M o l e c u l a r Bas is o f Ant igen Recogni t ion

Wayne Hein

S 6.1 DEVELOPMENT OF THE IMMUNOGLOBULIN REPER- TOIRE IN SHEEP

Reynaud, C-A.1; Hein, W.R.2; Dudler, L.2; Garcia, C.t; Marin, F. 1 and Weill, J-C. 1

llnstitut Necker, Facult6 de M6decine Necker, Enfants malades, 156 rue de Vaugirard, 75730 Paris Cedexl5 (France) and 2Basel Institute for Immunology, 487 Grenzacherstrasse, CH 4005 Basel (Switzerland)

Ileal Peyer's patches (IPP) are a primary site of B cell formation in sheep. We previously described that the immunoglobulin light chain genes are diversified during a several month period of development, starting in the late fetus, by a random somatic mutation mechanism, the mutants produced being submitted to an intense selection process. We have tried to investigate the nature of this selection by analyzing the immunoglobulin light chain repertoire in IPP in three different experimental conditions: in sheep thymectomized in the embryo (60 days of development); in a segment of the IPP isolated from the intestinal tract at the day 110 fetal stage; and lastly, in germ-free animals. The results obtained in these various conditions will be discussed.

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S 6 . 2 DIVERSITY OF THE CELL RECEPTOR REPERTOIRE IN SHEEP

Hein, W.R.

Basel Institute for Immunology, Basel, Switzerland

The relative prevalance of the two major T cell lineages in ruminants is unusual as compared to most other mammals since sheep and cattle have a large proportion of y~ T cells in their peripheral immune system. To determine whether these quantitative differences are accompanied by any qualitative features that might distinguish the level of receptor diversity present in ruminants, a number of molecular cloning techniques have been used to analyse the repertoire of sheep T cells. As in other species, sheep ~,c~ T cells generate a diverse repertoire of antigen receptors by rearrangement of many non- contiguous genes encoding V,D and J segments. The characterization of these genes is only at an early stage but it appears likely that the potential level of receptor diversity present in this population will be comparable to that in humans and mice. In contrast, sheep ~,c~ T cells have a much more extensive repertoire than their homologues in humans and mice, due to higher numbers of both variable and constant region genes. One family of V6 genes in particular has undergone extensive duplication. Moreover, hypervariable segments analogous to complementarity determining regions ( C D R ' s ) are readily indentifiable in the Vc~ sequences, suggesting that the germline repertoire has been shaped by intense operating at the level of ligand recognition. The Vy sequences on the other hand have been less extensively duplicated and show a different localisation of CDR's. The sheep 7c~ TCR occurs in a number of isotopic forms resulting from the association of a single C_3 segment with one of five Cy segments, each ofwich has distinctive structural features. The utilization of this expanded number of gene segments in peripheral ~,6 T cells is developmentally regulated and dependent on the continued presence of a functional thymus. The implications of these findings will be discussed in terms of the likely antigen recognition potential of sheep T cells.

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S 6 . 3 FUNCTIONAL STUDIES ON THE ROLE OF BOVINE MHC IN IMMUNE RECOGNITION

Letesson, J.J. and Lambot, M.

Immunology Unit FUNDP Namur Belgium

In spite of considerable progress made in the characterization of the restriction products of the BoLA system, very few informations are available about the importance of MHC polymorphism in antigen recognition and on the functional aspect of MHC-peptides and T cell receptors interactions in cattle.

However this kind of data appears to be of major concern in the field of subunit vaccine design.

On the contrary, in man and mouse species, very significant advances have recently been made in the understanding of "how MHC molecules work". Amongst the highlights of the last two years one can point out: isolation of pure MHC-peptides complexes, definition of stringent size requirements for peptides MHC class I interaction, X-ray crystallographic structure of the MHC class I bound peptides. Important advances have also been made in defining the role of /5 2 microglobulin in peptides-MHC interaction, the role of the invariant chain in MHC class II sorting, -interaction with peptides and the impact of peptides on MHC class II folding as well.

An overall wiew of the emerging picture of antigen processing and presentation will be shortly reviewed and illustrated with some data obtained from cattle.

With this in mind, selected studies unravelling how differences in MHC types can affect immune responses in cattle will be presented.

With respect to the BoLA class I molecules, despite increasing amount of data concerning the polymorphism (up to 50 specificities internationally recognized at two loci) their role in the restriction of T cell response has been only marginally studied. Godderis et al. made a pionneer work in the field with the study of cytotoxic T cell clones specific for Theileria Parva. They described more recently how the phenotype of restricting class I molecules can affect the strain specificity of the T cell clones.

On the MHC class II side, the lack of a suitable typing method for BoLA class II haplotype has been the major drawback limiting their functional study untill the development of 1D IEF typing. Since then, their influence on the T cell response has been illustrated by Glass and coworkers both for a multiepitopic antigen (ovalbumin) and for FMDV peptides.

In a more restrictive model we are currently involved in defining the minimal requirements for class II restricted T cell recognition in cattle by using

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purified class II molecules incorporated in artificial membranes. Data will be presented by illustrating an allogenic T cell recognition and a response to a superantigen drived by liposomes bearing purified bovine class II molecules.

Goddeeris et al; PNAS (1986) 83, 5238 Goddeeris et al; Immunol (1990) 69, 38 Glass et al; Immunol (1991) 74, 594 Glass et al; Immunol (1991) 72, 380