Allorecognition, defined as the ability to discriminate between self and non-self, is ubiquitous to colonial metazoans and widespread in aclonal taxa. Invertebrate allorecognition phenomena are of broad interest and have long captured the attention of geneticists by virtue of the allotypic diversity they display, marine ecologists by virtue of their control of effector mechanisms determining the outcome of intraspecific competition, evolutionary biologists by virtue of their regulation of the level at which selection acts, and immunologists by virtue of their resemblance to the allogeneic interactions that characterize pregnancy and transplantation in vertebrates. Diverse histocompatibility modes have been described in the jawed vertebrates, protochordates, and cnidarians, which are to date the only three taxa for which a genetic model to study allorecognition has been developed. Outside of the MHC-based histocompatibility of vertebrates, allorecognition determinants have been recognized in only two invertebrates. In the tunicate Botryllus, two genes involved in the histocompatibility response were characterized, FuHc and fester. In Hydractinia, the loci controlling allorecognition, alr1 and alr2, were mapped to a single chromosomal region, the allorecognition complex, and alr2 was recently identified as a polymorphic immunoglobulin superfamily (IgSF) receptor. In this study, the identification of the second Hydractinia allodeterminant, alr1, was undertaken. Chapter I briefly reviews prominent allorecognition model organisms and details the phenomenon in the model organism, Hydractinia symbiolongicarpus studied here. Chapter II describes the isolation of a 300.8 kb alr1-containing chromosomal interval by positional cloning. The analysis of that interval for its gene content and the determination of a primary alr1 candidate, CDS4, are described in Chapter III. Chapter III also reveals the existence of a complex of IgSF-like genes, to which belongs CDS4. CDS4, a novel polymorphic IgSF receptor that encodes a type I transmembrane protein with two hypervariable immunoglobulin-like extracellular domains, was confirmed to be the alr1 allodeterminant in Chapter IV, based on the investigation of natural polymorphism. CDS4 allele sequences were found to largely predict the outcome of allorecognition responses within and between laboratory lines and wild-type colonies, confirming the identity of CDS4 as the classically defined locus alr1.

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Solid organ transplantation is now an established and effective treatment option for end-stage organ failure. Whilst early outcomes have improved significantly over recent decades, longer-term outcomes have changed little. Despite advances in immunosuppression, most transplanted organs suffer an inevitable decline in function attributed to chronic rejection. It is evident that the alloimmune response remains incompletely characterised. Crucially, despite description several decades ago, the precise contribution that the direct (recognition of intact allogeneic MHC) and indirect (recognition of self-MHC restricted allopeptide) pathways make to allograft rejection remains incompletely understood. In this thesis, murine models of heterotopic cardiac transplantation have been utilised to analyse these pathways. The key findings of this work are as follows: 1) If able to evade NK cell killing, passenger donor CD4 T cells can make cognate, direct-pathway, interactions with recipient B cells. This interaction results in augmentation of all arms of the alloimmune response and acceleration of allograft rejection. 2) Direct-pathway CD4 T cell allorecognition is restricted to the immediate post transplantation period. Donor APCs are the major source of MHC class II for direct-pathway priming, and these are cleared rapidly by both innate and adaptive responses of the recipient, effectively limiting the longevity of direct allorecognition. 3) The duration of indirect-pathway responses against different alloantigens is variable, limited by availability of donor antigen. Expression of donor MHC class II is restricted to APCs and possibly endothelium (where expression is transient) limiting the duration of indirect-pathway allorecognition against MHC class II alloantigen. Indirect-pathway CD4 T cell responses targeted against parenchymal alloantigen are long-lived, and can provide help for generating alloantibody against different MHC alloantigens. 4) In response to continual presentation of target epitope indirect-pathway CD4 T cell responses against parenchymal expressed alloantigen are long-lived. The continual division of these cells results in greatly increased numbers of alloantigen-specific CD4 T cells in the chronic phase of the response, but despite this, memory responses are impaired. 5) Generating indirect-pathway regulatory T cells specific for parenchymal expressed alloantigen appears to be the most effective strategy to ameliorating chronic rejection.

All animals have evolved mechanisms to recognise and eliminate nonself in order to defend against invading pathogens and to prevent chimerism, the fusion between genetically distinct conspecifics. Like other metazoans, sponges are known to rely on sophisticated systems to maintain their self-integrity. As poriferans are also considered one of the most ancient extant metazoan phyla, they represent a critical comparative model for understanding the early evolution of immunity and self/nonself recognition in animals. The Toll-like receptor (TLR) signalling cascade plays a crucial role in immunity, and recent findings in the sponge Suberites domuncula suggest that its origin could predate eumetazoan cladogenesis. My genome and expressed sequence tag (EST) screens of the demosponge Amphimedon queenslandica detected homologues to most components of this pathway, supporting the notion that a primordial TLR signalling cascade emerged at the dawn of the Metazoa. The sponge also encodes a couple of putative TLR-related proteins (AmqIgTIRs) that consist of at least one extracellular immunoglobulin (Ig) and an intracellular Toll/Interleukin-1 receptor/resistance (TIR) domain. The presence of other unconventional TLRs in S. domuncula and in cnidarian representatives, implies that an ancestral TLR probably existed in the last common ancestor of all living metazoans, and independent duplication and divergence events led to the variety of forms observed in animals. Among the putative transcription factors present in Amphimedon, which are known to be activated by the TLR signalling cascade in other eumetazoans, I detected a single member of the Rel/nuclear factor-kappaB (NF-κB) family, AmqNF-κB, which is also the only Rel homology domain (RHD)-containing gene present in the sponge. This gene encodes a protein that is equipped with both a RHD and ankyrin (ANK) repeats, suggesting that the ancestral metazoan NF-κB was configured identically to contemporary vertebrate and sponge forms, and that the truncated NF-κB found in Nematostella vectensis resulted from the secondary loss of ANK. Aside from immunity, the Toll and TLR pathways contribute to a variety of biological processes in bilaterians, however their functions have only been investigated in detail in a limited number of metazoan model organisms. While studies have tested the immune role of various sponge genes, including components of the TLR cascade, no research has yet established whether they are also involved in development. Therefore, I investigated the expression of some of the immunity-related genes I isolated in Amphimedon in a developmental and immune context to shed light on the potential ancestral function(s) of the proteins they encode. Using in situ hybridisation, I demonstrate that AmqIgTIR2, AmqMyD88, AmqTollip, AmqPellino and AmqNF-ĸB are expressed during A. queenslandica early development. In contrast, the spatial and temporal expression of AmqIgTIR1 suggests it might encode a receptor that is specifically involved in the detection of metamorphic cues in larvae. A real-time quantitative PCR (qPCR) study performed on a pool of adult sponge cDNAs indicates that the expression levels of AmqIgTIR1, AmqIgTIR2, AmqMyD88 and AmqTollip are significantly affected by a nine-hour incubation in 50 µg/ml of lipopolysaccharide (LPS), and to a lesser extent by 105 colony forming units (cfu)/ml of live Vibrio harveyi. The gene expression of AmqIgTIR1 and AmqIgTIR2 suggests that they may encode proteins with antagonistic immunological functions. While AmqPellino and AmqNF-ĸB do not appear to be affected by LPS and Vibrio exposure, it is possible that these genes do not participate in the early immune response of poriferans. Together, my data indicate that the sponge genes surveyed might encode proteins that perform developmental, sensory and immunological functions, suggesting their roles could have also been multifaceted in the last common ancestor to all living metazoans. As is observed in other invertebrates, poriferans display an ontogenic shift in allorecognition; genetically different individuals can fuse during early development but, in most instances, not as adults. However, there is a limited understanding of the cellular organisation of sponge chimeras and the onset of poriferan allorecognition response. By following the fates of fluorescently tagged cells derived from genetically distinct Amphimedon larvae that are fused together at metamorphosis, I establish that there is a rapid ontogenic shift in the sponge allogeneic response about two weeks after the initiation of metamorphosis. Moreover, the molecular basis of the poriferan allorecognition system is possibly involved in creating differential cell affinities, which underlie the construction of the sponge body plan. Compatible with this scenario is the observation that cells from postlarvae that are allowed to develop for two weeks before contact do not fuse, and form a distinct boundary between genotypes. The molecules responsible for sponge cell reaggregation, the aggregation factors (AFs), have been proposed to drive the allorecognition response in poriferans. Notably, the Amphimedon genome encodes six putative AFs, of which five occur in a cluster. These findings indicate that the polymorphic variation observed in other poriferan AFs is probably the result of allelic variations of multiple genes belonging to the same family.

Allorecognition, defined as the ability to discriminate between self and non-self, is ubiquitous to colonial metazoans and widespread in aclonal taxa. Invertebrate allorecognition phenomena are of broad interest and have long captured the attention of geneticists by virtue of the allotypic diversity they display, marine ecologists by virtue of their control of effector mechanisms determining the outcome of intraspecific competition, evolutionary biologists by virtue of their regulation of the level at which selection acts, and immunologists by virtue of their resemblance to the allogeneic interactions that characterize pregnancy and transplantation in vertebrates. Diverse histocompatibility modes have been described in the jawed vertebrates, protochordates, and cnidarians, which are to date the only three taxa for which a genetic model to study allorecognition has been developed. Outside of the MHC-based histocompatibility of vertebrates, allorecognition determinants have been recognized in only two invertebrates. In the tunicate Botryllus, two genes involved in the histocompatibility response were characterized, FuHc and fester. In Hydractinia, the loci controlling allorecognition, alr1 and alr2, were mapped to a single chromosomal region, the allorecognition complex, and alr2 was recently identified as a polymorphic immunoglobulin superfamily (IgSF) receptor. In this study, the identification of the second Hydractinia allodeterminant, alr1, was undertaken. Chapter I briefly reviews prominent allorecognition model organisms and details the phenomenon in the model organism, Hydractinia symbiolongicarpus studied here. Chapter II describes the isolation of a 300.8 kb alr1-containing chromosomal interval by positional cloning. The analysis of that interval for its gene content and the determination of a primary alr1 candidate, CDS4, are described in Chapter III. Chapter III also reveals the existence of a complex of IgSF-like genes, to which belongs CDS4. CDS4, a novel polymorphic IgSF receptor that encodes a type I transmembrane protein with two hypervariable immunoglobulin-like extracellular domains, was confirmed to be the alr1 allodeterminant in Chapter IV, based on the investigation of natural polymorphism. CDS4 allele sequences were found to largely predict the outcome of allorecognition responses within and between laboratory lines and wild-type colonies, confirming the identity of CDS4 as the classically defined locus alr1. / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished