Expression of Major histocompatibility complex genes in carp (Cyprinus carpio L.)

The common carp ( Cyprinus carpio L.) has been the experimental animal of choice because many features of the immune system of this Cyprinid fish have been well characterized. The immune system consists of an integrated set of organs containing cells such as Ig + B lymphocytes, Ig -leucocytes, and macrophages, capable of performing a specific immune response. The prerequisites for such a response upon an antigenic challenge are cell-surface molecules, like T- and B-cell receptors, and major histocompatibility complex (MHC)-encoded molecules. T cells are only capable of recognizing processed antigen when it is presented in the context of an MHC molecules. In mammals it has been firmly established that peptides derived from the antigenic proteins are bound to MHC-encoded molecules, and that the peptide/MHC configuration is recognized by the T-cell receptor (TCR). Thus, for a better understanding of the initiation of a specific immune response more knowledge is needed about the presence and function of the molecules involved in antigen presentation.In Chapter 1 , a description is given of the current knowledge on the MHC in fish, and in particular in the common carp. The first MHC genes were described for carp, however, these were only partial genomic sequences, and it proved difficult to establish that these were functional genes. This first report was soon followed by a wealth of other sequences in a variety of other teleost species. Overall, the MHC gene structure and that of the beta 2 -microglobulin (β 2 m) do not seem to differ from those described for mammalian species. The MHC genes show an exon and intron structure remarkably similar to their mammalian counterparts, including the fact that the introns are all phase 1. The only major difference may lie in the fact that teleost fish have more than one MHC, similar to the situation described for the chicken and Xenopus MHC.Most sequences reported are, however, partial sequences obtained by Polymerase Chain Reactions (PCR) on genomic DNA, and do not provide information on the function of the encoded molecules. For a limited number of species, including the carp, full-length cDNA sequences have been reported, and can be used to infer the functionality of the encoded proteins. Analyses of these cDNAs have indicated that invariably the main functional characteristics, such as the presence of conserved peptide-binding residues and cysteines forming disulphide bridges, are present. Thus, although abundant theoretical evidence seems to suggest the presence of MHC molecules, formal proof has yet to be presented.In Chapter 2 , studies are described which aim at providing evidence for the the presence of MHC class II molecules in lymphoid organs. To this end RNA was isolated from several organs, some with known immunological functions. The cDNA prepared from it was used as a template in the PCR amplification of MHCCyca-DAB transcripts. The presence of these transcripts appeared to be confined to tissues such as thymus, spleen, pronephros and intestine, which have been demonstrated to perform immunological functions. Further analyses carried out on isolated leucocyte subpopulations indicated that a direct correlation exists between the levels of Cyca-DAB expression and the number of Ig +cells present. In addition, adherent cells were shown to abundantly express class II transcripts. The most important finding was the fact that thymocytes were the cell population with the highest expression of Cyca-DAB mRNA. Although we were unable to detect the MHC class II molecules themselves due to the lack of proper reagents like antibodies, these studies reinforced the notion that class II expression is restricted to those microenvironments where antigen presentation takes place.To overcome the problem of detection of the molecules proper, a different strategy was adopted, i.e. , prokaryotic expression of cDNA sequences for the production of recombinant proteins that can be used to immunize rabbits. In Chapter 3 , experiments are described with a polyclonal antibody raised against carp β 2 -microglobulin (Cyca-B2m). This antiserurn was used to assess the expression of class I molecules on the cell surface of different cell populations. The results of these experiment show that erythrocytes and thrombocytes are negative, whereas leucocytes of lympho-myeloid lineages are class I positive. In addition, a brightly class I positive population of Ig -lymphocytes was identified, which may constitute putative circulating T lymphocytes.Subsequently, experiments were designed to study the effect of temperature on the expression of class I molecules on peripheral blood leucocytes (PBL). These experiments revealed a long lasting absence of class I molecules at low permissive (6°C) temperatures, which could be restored by increasing the temperature. These results were confirmed by using an antiserum raised against the carp class I αchain (Cyca-UA). However, the presence of Ig on the cell surface of B cells remained unchanged in the course of the experiments. The transcription of the genes involved was also studied, using PCR amplification on cDNA prepared from RNA. Normal transcription of Cyca-UA was observed, which contrasts the low levels of transcription found for Cyca-B2m. Therefore, the absence of class I molecules is considered to be the result of a lack of sufficient Cyca-B2m. transcription, prompting the conclusion that class I cell surface expression is regulated by a temperature sensitive transcription-mechanism of the β 2 m gene.In Chapters 4 and 5 , the MHC class I and class II molecules were studied during carp ontogeny using different approaches. In earlier stages of development, studies on the expression of the MHC class I and class II molecules have been restricted to the detection of transcripts using PCR amplification of cDNA. In later developmental stages, where it was possible to obtain cell suspensions from immunological organs of the larvae, the expression of MHC class I molecules was studied by using polyclonal antibodies to β 2 m and the MHC class I αchain. In unfertilized eggs no transcription of any of the genes was detected. Transcription of Cyca-UA, Cyca-DAB and Cyca-DXA starts as early as day 1, and increases steadily reaching a plateau at day 3. In contrast, transcription of Cyca-B2m was shown to start at day 7. After 14 days, the levels of expression of the genes under investigation have reached a plateau. At this point in time, organs can be dissected and used for the detection of transcription. These experiments demonstrated that from the lymphoid organs investigated, the spleen is the only one where a significant lower level of transcription of the MHC and β 2 m genes was found. This observation correlats with the late development of this organ and subsequent late influx of lymphoid cells. The absence of Cyca-B2m transcripts suggests a lack of class I cell surface expression, similar to the situation in the temperature experiments, and corroborates the conclusion that AMC class I molecules do not play a major role during early ontogeny.At three weeks after fertilization it is possible to obtain enough cells from the immunologically important organs to perform FACS analyses with antibodies identifying MHC class I molecules. These studies revealed that, in the pronephros and spleen, cells are present, up to week 13, which are positive for Cyca-B2m, but do not express the Cyca-UA class I a chain. This cell population seems to consist mainly of Ig +cells. No difference in the percentage of Cyca-B2m- and Cyca-UA-positive cells is observed after week 13, reflecting the adult situation. In the adult thymus, to the contrary, there remains a population of thymocytes which is Cyca-B2m-positive, but Cyca-UA-negative. The identity of the class I αchain, that is associated with the Cyca-B2m during ontogeny and in the adult thymus, is yet to be revealed. The suggestion is that a non-classical class I-like molecule may play a role in thymocyte differentation. In the adult carp, peripheral blood consists of Cyca-UA- and Cyca- B2m-negative erythrocytes and thrombocytes, whereas the other leucocytes are positive for these molecules.In Chapter 6 , the data presented in this thesis are discussed in connection with what is known from other vertebrates, mainly Xenopus and chicken. The MHC class I and class II molecules are dealt with separately, as their distribution patterns differ to a large extent. Basically, the expression pattern of class II molecules follows that of other species studied. The only exception is the early onset, in carp, of MHC class II transcription during ontogeny. As for the expression of class I molecules on cells of the immune system it is discussed that different class I αchain-encoding genes are being used in carp. This conclusion is based on the observations from experiments with the antiserum to the Cyca-B2m molecule. However, the most interesting finding, with respect to the MHC class I expression, is the temperaturedependent regulation of β 2 m transcription. This mechanism is thought to be responsible for the lack of MHC class I cell surface expression that has been observed at low ambient temperatures in this ectothermic vertebrate species. This is the first report in a cold-blooded vertebrate in which this mechanism has been firmly established.