Molecular analysis of ovine CD1 expression

The CD1 family is a family of molecules with structural homology to MHC class I and low but significant sequence homology to both MHC class I and MHC class II. A key feature of the CD1 family is restricted tissue distribution. This distribution varies with isotype but includes cortical thymocytes, antigen presenting cells and intestinal epithelial cells. It has recently been shown that CD1 can present mycobacterial lipid antigens to T cells (Beckman et al. 1994, Sieling et al. 1995). Another major feature of CD1 is limited polymorphism which is in contrast to the extensive polymorphism associated with MHC molecules.

In humans, five CD1 genes exist (CD1A-E) which encode four CD1 molecules (CDla-d). CD1 genes have also been described in mice, rats, rabbits and sheep. Studies in cattle and pigs have been at the protein level using anti-CD 1 mAbs. Studies on ovine CD1 were initially carried out using mAbs to study expression in cells and tissues. More recently, four ovine CD1 cDNA clones (SCD1A25, SCD1B42, SCD1B52 and SCD1T10) have been isolated all of which have closest homology to human CD IB (Ferguson et al. 1996). SCD1B52 contains a precise deletion of exon 4 which encodes the oc3 domain. Southern hybridization has indicated the existence of up to seven ovine CD1 genes.

The aim of the work carried out in this thesis was to further investigate the ovine CD1 family and clarify the existing information at the cellular and molecular level. Initial studies utilised existing anti-CD 1 mAbs to clarify the pattern of tissue expression of ovine CD1. Two distinct clusters of mAbs were shown to exist - the majority recognise a molecule with tissue distribution similar to CD lb whilst three mAbs, SBU-T6, CC43 and CC118 demonstrate staining of tissue macrophages, the majority of B cells and monocytes in addition to thymocytes and dendritic cells. NH2-terminal sequencing was subsequently used to establish the antigens recognised by the mAbs SBU-T6 and CC14. This technique demonstrated that the CC14 antigen was consistent with the predicted sequence of the SCD1B42 cDNA clone whereas the SBU-T6 antigen had closest homology to the predicted amino-acid sequence of the human CD IE gene. This is particularly noteworthy as no protein product of the CD IE gene has yet been described in any species.

Subsequent work attempted to isolate the gene encoding the molecule recognised by SBU-T6 using a transient expression system in which COS cells were transfected with a lymph node cDNA library contained within the vector pcDNA3. This was unsuccessful, however a sheep CD ID-like sequence was isolated from this library utilising primers based on the NH2-terminal sequence of the SBU-T6 antigen. Expression of the SCD1D gene was investigated using in situ hybridization and RT-PCR. SCD1D transcripts were demonstrated in thymus, liver, intestine, lymph node and PBLs. A further experiment investigated the expression of the SCD1B52 gene (which contains a precise deletion of exon 4). These studies have extended the knowledge of the ovine CD1 family and establish it as one of the most complex described to date. This work has demonstrated that sheep clearly express multiple CD1 isotypes as in man and rabbits in addition to the multiple CD IB-like genes reported previously.