REVIEW

René Stet’s impact on the study of teleost majorhistocompatibility genes: evolution from loci to populations

Brian Dixon

Received: 17 December 2007 /Accepted: 19 December 2007 / Published online: 10 January 2008# Springer-Verlag 2007

Abstract René Josephus Maria Stet pursued a 35-year-long scientific career contributing to human immunology,shrimp immunity and teleost immunity. His most signifi-cant contributions, however, were to the field of teleostmajor histocompatibility (MH) gene research from 1988 to2007, a field in which he was a leader and an innovator.This review will discuss his work on these genes, high-lighting the impact he had in three temporally overlappingphases of his career that can be characterized as MH genediscovery, MH gene function and evolution and populationdynamics of teleost MH genes.

Keywords Major histocompatibility . Teleost . Evolution .

Populations

Introduction

Before discussing René’s many research contributions, it isimportant to note that he was a man of ideas. He was notshy about expounding his thoughts, and he published atleast nine insightful reviews, always with a central idea toshare, usually with a trademark witty subtitle separated by acolon (hence the title of this review). One of his morecritical ideas was the fact that, given the lack of linkage ofmajor histocompatibility (MH) genes in teleosts, theirnomenclature needed to be differentiated from the termmajor histocompatibility complex (MHC) used for all other

vertebrates, in which these genes are linked (Flajnik andKasahara 2001). His first musings on this were shown inthe title of his abstract for the Fifth International Workshopon MHC Evolution in Visby, Sweden, which, based on hisstudies of the linkage of carp MH class I genes, referred tothe teleost unMHC (Stet et al. 1997). After the publicationof several studies clearly demonstrating the lack of linkageof these genes in most teleosts (Sato et al. 2000), Renéstarted referring to them as simply MH genes, as he felt the“complex” portion of the name was indicative of thelinkage of genes within the genetic complex found intetrapods and later in sharks (Ohta et al. 2000). He feltstrongly enough about this that we suggested dropping thecomplex for teleosts in a review article that we co-authoredin 2001 (Dixon and Stet 2001), and he vigorously defendedthis idea in our 2003 “Road Less Taken” review (Stet et al.2003). That review also included another idea René heldabout the evolution of teleost immune systems in general:That they had diverged from the line of evolution leading totetrapods, thus creating two parallel evolutionary histories,rather than representing a single evolutionary continuum.

MH gene discovery

René’s Ph.D. research was on graft vs host disease in rats(Stet et al. 1985, 1986, 1987; Nieuwenhuis et al. 1988; Atenet al. 1992), and after that, he spent a year in Scotlandstudying the genetic basis of disease in fish; hence, he wasperfectly placed to start researching MH genes in teleostswhen he arrived in Wageningen in 1988. At this time, teleostexperiments showing the presence of IgM in teleosts had beenpublished (Wetzel and Charlemagne 1985; Bly et al. 1986;),and allogeneic grafting experiments (Van Muiswinkel et al1986; Nakanishi 1987) and mixed leukocyte reactions (Caspi

Immunogenetics (2008) 60:77–82DOI 10.1007/s00251-007-0272-0

B. Dixon (*)Department of Biology, University of Waterloo,200 University Ave W,Waterloo, Ontario, Canada, N2L 3G1e-mail: bdixon@sciborg.uwaterloo.ca

and Avtalion 1984; Miller et al. 1986) had shown rejectionand alloreactivity in a manner consistent with those mediatedby MH genes in mammals, but MH proteins and genes hadnot yet been definitively identified in teleosts. Renéparticipated in some of this work (Kaastrup et al. 1989) thatidentified certain loci responsible for transplant rejection, buthis real innovation was to see the potential application ofmolecular biology in confirming the presence of these genesin teleosts. This skill, combined with the invaluable resourceof gynogenetically defined isogenic strains available inWageningen, meant René was able to take advantage ofHashimoto’s September 1990 groundbreaking report of carpsequences with sequence similarity to human MHC genes(Hashimoto et al. 1990). He rapidly used the probesHashimoto and co-workers had developed to show that theWageningen lines not only had bands that reacted bySouthern hybridization but also that the gynogenetic linesall had the same size reactive bands. Additionally, he isolateda cDNA clone using the MH class II probe. René reported allof this in the dramatic session on Genetic Aspects ofImmune Reactivity at the 5th International Society ofDevelopmental and Comparative Immunology congress inAugust 1991 (Stet et al. 1991), in which several groupsreported the cloning of cDNA clones to MH class II fromsalmon (Fosse et al. 1991) and trout (Glamann et al. 1991),and which also included my presentation of the degeneratepolymerase chain reaction (PCR) cloning of beta-2-micro-globulin from tilapia (Dixon et al. 1991). At that meetingRené, my supervisor Bill Pohajdak and I agreed tocollaborate in obtaining the carp beta-2-microglobulincDNA, resulting in the first report of beta-2-microglobulincloning from fish (Dixon et al. 1993) and heralding the startof a long and productive collaboration between René andmyself. René continued to carefully characterize all of theMH genes from carp over the next couple of years, notrushing to be first to publish gene sequences but carefullydefining each gene he cloned using the carp gynogeneticlines. He collaborated with Jan Klein’s group in the cloningof the MH class II beta gene (Ono et al. 1993), and hedefined the inheritance of these genes and the only knowncarp class I gene at that point, Cyca-Z, in the gynogeneticcarp system (Stet et al. 1993). René’s patient approach paidoff, when, in 1996, his Ph.D. student Saskia van Erppublished the carp classical MH class I locus, Cyca-UAA,and showed that the Cyca-Z lineage was non-classical (vanErp et al. 1996a, b, c). With the publication that year of apaper describing the inheritance of class II alpha and beta incarp (van Erp et al. 1996a, b, c), René had characterized allof the MH genes from carp, defining their inheritance withinthe gynogenetic carp lines to show that the class II betagenes were in two linked pairs, while the two class I geneswere unlinked loci. René continued to contribute to thecloning and characterization of MH genes from several

species over the years (Lundqvist et al. 1999; Kruiswijk et al.2002; Buonocore et al. 2007), including collaboration withLars Pilstom in which they would report that Atlantic codhad up to 34 expressed MH class loci (Persson et al. 1999).This is very unusual, because other vertebrates deletechromosomal regions to maintain a copy number of six orless (Courtet et al. 2001), which is thought to be optimal,because the expression of more that this number wouldeliminate so many T-cells during development that theindividual would have too few left to combat pathogens.René actually helped to confirm this observation later onwhen he studied barbels, a hexaploid cyprinid, and showedthat despite their polyploidy, they expressed only five class Iand four of both MH class II genes (Kruiswijk et al. 2004).This was yet another astonishing discovery from Atlanticcod, which has been shown to lack specific antibodyresponses (Magnadottir et al. 2001). René collaborated withUnni Grimholt in characterizing both salmonid class II(Grimholt et al. 2000) and class I loci (Grimholt et al. 2002),when he realized that salmon’s much wider use inaquaculture provided more funding and opportunities forresearch.

MH gene function

Naturally, once MH gene sequences were discovered inteleosts, the next step was to show that they were expressedand functioned in a manner consistent with their role inantigen presentation and self-recognition. René workedwith his Ph.D. student Pablo Rodrigues to study theexpression pattern of MH class II genes in carp tissuesand blood lymphocytes, showing that they there expressedin thymus, head kidney, spleen and gut and blood(Rodrigues et al. 1995). Blood lymphocytes with high-surface immunoglobulin were positive, as were adherentlymphocytes and thymocytes, a profile that should beexpected for MH class II. When I arrived in Wageningen, Ihad brought a polyclonal antiserum to carp beta-2-micro-globulin with me and master’s student Jeroen Roelofsworked with René and me to develop a polyclonalantiserum to carp MH class I. Pablo used these to furtherexamine the function of MH class I receptors in carp.

Firstly, we examined the effect of environmentaltemperature on MH gene expression. I had just written abook chapter discussing the “empty MHC class I”phenomenon (Pohajdak et al. 1993), which showed thatmammalian cells incubated at 10°C lower than normal bodytemperature and expressed class I MHC heavy chains withno peptide or beta-2 microglobulin associated with them(Ljunggren et al. 1990; Rock et al. 1991). Pedro, René and Idiscussed what happens in teleost fish cells, because asectotherms, they experience large changes in body temper-

78 Immunogenetics (2008) 60:77–82

ature, sometimes on a daily basis. Our working hypothesiswas that there would be no change in expression related totemperature in teleosts: Why would they leave themselvesopen to pathogens? The results of the experiment were,however, startling. While carp kept at 24°C had no changein expression of class I MH receptors on their bloodleukocytes and carp kept at 12°C showed a small transientdownregulation of these receptors, animals kept at 6°Ccompletely removed these receptors from the surface oftheir blood leukocytes (Rodrigues et al. 1998a, b). Exami-nation of gene expression by reverse transcriptase showedthat the MH class I heavy chain gene was expressed at alltemperatures, but that the beta-2 microglobulin gene wasspecifically turned off at 6°C (Rodrigues et al. 1998a, b).Thus, the carp had deliberately immunosuppressed them-selves at low temperatures, very probably as a method ofconserving energy, as they were not eating at these lowtemperatures. The only follow-up work on this, from mylab, was published last year, and it showed that whilerainbow trout turn off MH genes at low temperature, theyturn off MH class II gene transcription, not MH class I(Nath et al. 2006). These observations would not have beenpossible without René’s initial willingness to start this lineof research, and they show one of the many startlingdifferences that teleost immune systems have from theirmammalian equivalent, despite the fact that both containbasically the same sets of cells and genes.

The second functional study that René undertook, againwith Pablo Rodrigues as the lead, was to examine theinduction of MH gene expression during the ontogeny ofcarp (Rodrigues et al. 1998a, b). This study revealed thatwhen whole larvae are examined by reverse transcriptasePCR, while MH class II and class I heavy chain genes areturned on almost immediately after hatching and build tomaximum expression after 7 days, the b2m gene is notexpressed at all until day 7. Interestingly, however, in olderlarvae where lymphocytes from individual tissues could beexamined by flow cytometry, b2m expression in proneph-rocytes, thymocytes and B cells reached maximal levelsweeks before Cyca-UA, the classical MH class I gene, withboth being maximally expressed at 13 weeks post hatch.This suggested that larval fish would not have completeimmune function until that point.

Along the way, René’s lab had also shown that thaterythrocytes and thrombocytes were negative for MH classI (Rodrigues et al. 1998a, b; van Erp et al. 1996a, b, c).While the expression of MH class I on erythrocytes variesdepending on species, thrombocytes (platelets) are usuallypositive for these receptors, another key difference betweenmammals and teleosts. Thus, René Stet’s leadershippioneered studies on the function of MH molecules inteleosts, showing that while they are similar to mammalianimmune systems, they function very differently.

Evolution and population dynamics of teleost MH genes

For René, investigating the polymorphism and evolution ofteleost MH genes was initially another aspect of thefunctional characterization. As part of the characterizationof the first MH class I genes isolated from carp, René andhis Ph.D. student Saskia van Erp discovered that there arefour very ancient lineages of class I genes in teleost fish(van Erp et al. 1996a, b, c). This contrasted with mammalianMHC class I genes, where it was not possible to identifyhomologues between humans and mice. This was the firstobservation that teleost MH genes evolved in a mannerdifferent from mammalian paradigm.

In the years immediately after the first isolation of MH genesequences demonstrating that they were polymorphic was animportant part of showing that they were really the teleostequivalents of mammalian MHC genes. Because this was notpossible using the inbred lines of carp available to René inWageningen, he initiated a collaboration with FerdinandSibbing and his Ph.D. student, Leo Nagelkerke, who wereworking on a unique cyprinid species complex—the barbelsfound in Lake Tana, Ethiopia. This lake, which was probablyless than a million years old, contained 16 unique morphotypesof barbel, each apparently occupying a distinct ecologicalniche (Nagelkerke and Sibbing 1996; Nagelkerke et al. 1994),a unique model system for analyzing segregation andselection of MH genes. René and I studied MH class II betaalleles in our of these morphotypes, discovering 57 alleles injust 17 individuals—more polymorphism that is found inmost mammals (Dixon et al. 1996). The class II allelessegregated completely between the morphotypes, suggestinga rapid evolutionary rate for MH class II genes in cyprinids,again differing from the evolution of mammalian MHC classII genes, where allelic lineages from humans could be tracedback to prosimians despite over 85 million years ofdivergence (Bontrop et al. 1999). René continued theanalysis of MH gene evolution in the Lake Tana barbelswith his Ph.D. student, Corrine Kruiswijk, who analyzedseven more morphotypes for MH class II alleles and alsoanalyzed MH class I alleles in six morphotypes. While noneof the species shared MH class II alleles, MH class I alleleswere shared between many of the morphotypes (Kruiswijk etal. 2005), adding further confirmation that MH class I genesevolved slowly and MH class II genes evolved rapidly incyprinids. Because Peter Parham’s lab had reported the samephenomenon in salmonids, in collaboration with René in2001 (Shum et al. 2001), confirming René’s initial observa-tions, it appeared that these modes of MH gene evolutionapplied to all teleosts. René, in collaboration with BillJordan, did however show that, true to the punctuatedequilibrium model of evolution, rapid evolution of salmonidsMH class I genes can happen under the appropriate selectivepressures (Consuegra et al. 2005).

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The Lake Tana barbel project had led René to his truepassion, the evolution and population dynamics of MHgenes in teleosts. At the ISDCI in Cairns, Australia, in2000, he confided in me that he was no longer reallyinterested in molecular details of the teleost MH proteinfunction such as antigen presentation, but wanted to focusinstead on population-based studies of genes, demonstrat-ing the function of these genes directly. This led René to hisbest and most exciting contributions to MH gene research,studies using Atlantic salmon as a model. René collaborat-ed with Unni Grimholt to clone and characterize the lastMH gene, class II alpha (Grimholt et al. 2000), and thentogether they began to study breeding families of Norwe-gian aquaculture stocks. They characterized the haplotypesof both MH class I and class II present within thesefamilies, showing that in both cases there was only a singlemajor expressed gene (Grimholt et al. 2002; Stet et al.2002). This line of research led to the landmark 2003 papershowing that families possessing particular MH class Ialleles had greater or sometimes worse survival rates thanothers during viral pathogen exposure and that the samewas true for families with particular MH class II allelesduring bacterial pathogen exposure; moreover, some class Ialleles also had an effect on resistance or susceptibility tobacterial pathogens (Grimholt et al. 2003). Because of thefact that the two classes of genes are unlinked in teleosts,the resistance to the two classes of pathogens segregatedindependently (Grimholt et al. 2003). It had long beenthought that the association of specific MH alleles withresistance to specific pathogens should result in increasedfrequencies of alleles in some populations, such as theHLA-B53 allele in Africans living in areas with a largeprevalence of malaria (Gupta and Hill 1995; Hill et al.1992), but this association had not been previouslydemonstrated in any species as clearly as in René and UnniGrimholt’s study.

A similar landmark contribution from René, again incollaboration with Bill Jordan, appeared in the Proceedingsof the Royal Society (de Eyto et al. 2007). Natural selectionby pathogens is thought to maintain MH gene polymor-phism in wild populations, but this idea lacked concreteexamples in wild populations. René and his colleaguesreleased eggs with the haplotype frequencies expected fromparental crosses into a river and then analyzed the survivingindividuals 6 months later. While the MH class I and non-MH linked marker allele frequencies did not diverge fromthe parental ratios, the MH class II frequencies divergedsignificantly from the expected ratio. This demonstrateddirectly that natural selection by pathogens had shaped theMH class II alpha allele frequencies.

René Stet passed away at the pinnacle of his career, atthe point where he was using model systems to provideclear direct evidence of the principles of MH gene

evolution, not just in teleost but in general. He was rightlydeclared the King of Fish MH genes at the seventhInternational Symposium on Fish Immunology, organizedby the Nordic Society for Fish Immunology, and his skillhad been further recognized when he was asked by theEuropean Bioinformatics Institute Immuno Polymorphismdatabase to maintain the MH polymorphism database forsalmonids. René Stet helped to found the investigation ofteleost immunity using molecular techniques, and he had aprofound impact on the field over the last two decades. Hisinsight, perseverance and vision will be sorely missed.

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