Allorecognition and chimerism in an invertebrate model organism

The presence of highly specific histocompatibility reactions in colonial marine invertebrates that lack adaptive immune systems (such as the sponges, cnidarians, bryozoans and ascidians) provides a unique opportunity to investigate the evolutionary roots of allorecognition and to explore whether homologous innate recognition systems exist in vertebrates. Conspecific interactions among adult animals in these groups are regulated by highly specific allorecognition systems that restrict somatic fusion to self or close kin. In Hydractinia (Cnidaria:Hydrozoa), fusion/rejection responses are controlled by two linked genetic loci. Alleles at each locus are co-dominantly inherited. Colonies fuse if they share at least one haplotype, reject if they share no haplotypes, and display transitory fusion if they share only one allele in a haplotype—a pattern that echoes natural killer cell responses in mice and humans. Allorecognition in Hydractinia and other marine invertebrates serves as a safeguard against stem cell or germline parasitism thus, limiting chimerism to closely related individuals. These animals fail to become tolerant even if exposed during early development to cells from a histoincompatible individual. Detailed analysis of the structure and function of molecules responsible for allorecognition in basal marine invertebrates could provide clues to the innate mechanisms by which higher animals respond to organ and cell allografts, including embryonic tissues.Key words: allorecognition, chimerism, invertebrate, innate immune system     

Allorecognition and chimerism in an invertebrate model organism

The presence of highly specific histocompatibility reactions in colonial marine invertebrates that lack adaptive immune systems (such as the sponges, cnidarians, bryozoans, and ascidians) provides a unique opportunity to investigate the evolutionary roots of allorecognition and to explore whether homologous innate recognition systems exist in vertebrates. Conspecific interactions among adult animals in these groups are regulated by highly specific allorecognition systems that restrict somatic fusion to self or close kin. In Hydractinia (Cnidaria:Hydrozoa), fusion/rejection responses are controlled by two linked genetic loci. Alleles at each locus are co-dominantly inherited. Colonies fuse if they share at least one haplotype, reject if they share no haplotypes, and display transitory fusion if they share only one allele in a haplotype – a pattern that echoes natural killer cell responses in mice and humans. Allorecognition in Hydractinia and other marine invertebrates serves as a safeguard against stem cell or germline parasitism thus, limiting chimerism to closely related individuals. These animals fail to become tolerant even if exposed during early development to cells from a histoincompatible individual. Detailed analysis of the structure and function of molecules responsible for allorecognition in basal marine invertebrates could provide clues to the innate mechanisms by which higher animals respond to organ and cell allografts, including embryonic tissues.     

Model systems of invertebrate allorecognition

Nearly all colonial marine invertebrates are capable of allorecognition--the ability to distinguish between self and genetically distinct members of the same species. When two or more colonies grow into contact, they either reject each other and compete for the contested space or fuse and form a single, chimeric colony. The specificity of this response is conferred by genetic systems that restrict fusion to self and close kin. Two selective pressures, intraspecific spatial competition between whole colonies and competition between stem cells for access to the germline in fused chimeras, are thought to drive the evolution of extensive polymorphism at invertebrate allorecognition loci. After decades of study, genes controlling allorecognition have been identified in two model systems, the protochordate Botryllus schlosseri and the cnidarian Hydractinia symbiolongicarpus. In both species, allorecognition specificity is determined by highly polymorphic cell-surface molecules, encoded by the fuhc and fester genes in Botryllus, and by the alr1 and alr2 genes in Hydractinia. Here we review allorecognition phenomena in both systems, summarizing recent molecular advances, comparing and contrasting the life history traits that shape the evolution of these distinct allorecognition systems, and highlighting questions that remain open in the field.     

Allorecognition polymorphism versus parasitic stem cells

Allorecognition--the ability of an individual to distinguish between self and non-self cells and tissues--ultimately depends on the presence of highly polymorphic gene(s). Allorecognition loci are the most diverse ever described, with tens to hundreds of alleles observed in a population. An unresolved problem in population genetics lies in understanding the origins, accumulation and maintenance of this extensive polymorphism, often over millions of years and across multiple speciation events. Botryllus schlosseri, a primitive chordate, has a life history that links several components of allorecognition from disparate fields that are experimentally accessible. This review outlines these traits and discusses some of the puzzling aspects of allorecognition in Botryllus that might contribute to understanding the evolution of these extraordinary polymorphisms.     

EVIDENCE FOR SELECTION ON A CHORDATE HISTOCOMPATIBILITY LOCUS

Allorecognition is the ability of an organism to differentiate self or close relatives from unrelated individuals. The best known applications of allorecognition are the prevention of inbreeding in hermaphroditic species (e.g., the self‐incompatibility [SI] systems in plants), the vertebrate immune response to foreign antigens mediated by MHC loci, and somatic fusion, where two genetically independent individuals physically join to become a chimera. In the few model systems where the loci governing allorecognition outcomes have been identified, the corresponding proteins have exhibited exceptional polymorphism. But information about the evolution of this polymorphism outside MHC is limited. We address this subject in the ascidian Botryllus schlosseri, where allorecognition outcomes are determined by a single locus, called FuHC (Fusion/HistoCompatibility). Molecular variation in FuHC is distributed almost entirely within populations, with very little evidence for differentiation among different populations. Mutation plays a larger role than recombination in the creation of FuHC polymorphism. A selection statistic, neutrality tests, and distribution of variation within and among different populations all provide evidence for selection acting on FuHC, but are not in agreement as to whether the selection is balancing or directional.     

Physiologically-Mediated Self/Non-Self Root Discrimination in Trifolium repens has Mixed Effects on Plant Performance

Recent studies suggest that plant roots can avoid competition with other roots of the same plant, but the mechanism behind this behavior is yet largely unclear and their effects on plant performance hardly studied. We grew combinations of two ramets of Trifolium repens in a single pot that were either intact, disconnected for a shorter or longer time, or that belonged to different genotypes. Interconnected ramets developed lower root length and mass than any other combination of ramets, supporting the notion that self/non-self discrimination in T. repens was based entirely on physiological coordination between different roots that develop on the same plant, rather than biochemical allorecognition. These responses were consistent among eight field-collected genotypes, suggesting that self/non-self discrimination is a common feature in wild populations of white clover. There were no significant treatment x genotype interactions suggesting that genetic variation for self/non-self discrimination may be limited. Self-interactions resulted in lower to similar shoot biomass and number of ramets, but higher flowering probabilities, compared to non-self interactions. Thus, our results demonstrated that the performance consequences of self/non-self discrimination may be more complicated than previously thought.Key Words: biomass allocation, clonal plants, competition, flowering, phenotypic plasticity, physiological coordination, plant growth, Trifolium repens, self/non-self discrimination     

Allorecognition mechanisms during ascidian fertilization

Ascidians (primitive chordates) are hermaphroditic animals, releasing sperm and eggs nearly simultaneously. But, many ascidians, including Ciona intestinalis and Halocynthia roretzi, show self-sterility or preference for cross-fertilization rather than self-fertilization. The molecular mechanisms underlying this allorecognition process are only poorly understood. We recently identified the genes responsible for self-incompatibility in C. intestinalis by a positional cloning: sperm-borne polycystin 1-like receptor, referred to as s-Themis, and its fibrinogen-like ligand called v-Themis on the vitelline coat (VC) are highly polymorphic and appear to be responsible for allorecognition in the fertilization of C. intestinalis. In H. roretzi, on the other hand, we revealed that HrVC70, a 70-kDa main component of the VC consisting of 12 epidermal-growth-factor (EGF)-like repeats, is a candidate allorecognition protein, since the attachment of this protein to the VC during oocyte maturation and its detachment by weak acid are closely linked to the gain and the loss of self-sterility, respectively, and also since nonself-sperm rather than self-sperm efficiently bound to HrVC70-agarose. As a binding partner of HrVC70, a 35-kDa GPI-anchored glycoprotein in sperm lipid rafts, referred to as HrUrabin, was identified: HrUrabin appears to play a key role in allorecognizable sperm binding to HrVC70 during fertilization. In the present review, we describe the current progress on the molecular bases of allorecognition, or self-incompatibility, during ascidian fertilization, by considering the SI systems in another organisms including fungies and flowering plants.     

Allorecognition and xenorecognition in reef corals: a decade of interactions

Xenorecognition phenomena in coral reefs are expressed by a striking array of morphological and cytological responses. Corals encountering conspecifics further elicit additional repertoires of effector mechanisms, specific to allogeneic challenges. Both inducible sets of antagonistic machineries of allo- and xenoresponses are highly specific. In many cases, they are predictable, reproducible, and reveal the hallmark of coral tissue capacity to distinguish between self and non-self. This essay summarizes a decade (1992 –2002) of published results on reef coral immune features. While studies on xenorecognition uncovered the existence of established, non-transitive hierarchies and the importance of antibacterial/cytotoxic compounds secreted by corals, allorecognition assays disclosed the presence of specific and complex non-transitive hierarchies dictated by the expression of a variety of effector mechanisms (‘tailored’ against different conspecifics), the existence of gradual maturation of alloresponses (important in the formation of natural chimeras), the debatable issue of allorecognition memory, and the appearance of delayed, second sets of alloresponses. A critical evaluation of historecognition reveals that expressed responses in different coral systems are phenotypically matched with counterpart outcomes recorded in the mammalian immune systems. Histocompatibility in corals, as in vertebrates, relies on recognition elements (not yet disclosed on the molecular level) as well as on effector mechanisms.     

Self/non-self discrimination in angiosperm self-incompatibility

Self-incompatibility (SI) in angiosperms prevents inbreeding and promotes outcrossing to generate genetic diversity. In many angiosperms, self/non-self recognition in SI is accomplished by male-specificity and female-specificity determinants (S-determinants), encoded at the S-locus. Recent studies using genetic, molecular biological and biochemical approaches have revealed that angiosperms utilize diverse self/non-self discrimination systems, which can be classified into two fundamentally different systems, self-recognition and non-self recognition systems. The self-recognition system, adopted by Brassicaceae and Papaveraceae, depends on a specific interaction between male and female S-determinants derived from the same S-haplotype. The non-self recognition system, found in Solanaceae, depends on non-self (different S-haplotype)-specific interaction between male and female S-determinants, and the male S-determinant genes are duplicated to recognize diverse non-self female S-determinants.     

How a home-grown T cell receptor interacts with a foreign landscape

The ability of a T cell receptor (TCR) to directly recognize foreign (allogeneic)-major histocompatibility complex (MHC) molecules underlies T cell-mediated rejection in patients receiving allogeneic organ transplants. In this issue, Colf et al. (2007) reveal that instead of mimicking the interactions formed with a self MHC, a single TCR adopts a completely different strategy to recognize a foreign MHC.     

Self-recognition affects plant communication and defense

Animals have the ability to distinguish self from non-self, which has allowed them to evolve immune systems and, in some instances, to act preferentially towards individuals that are genetically identical or related. Self-recognition is less well known for plants, although recent work indicates that physically connected roots recognize self and reduce competitive interactions. Sagebrush uses volatile cues emitted by clipped branches of self or different neighbours to increase resistance to herbivory. Here, we show that plants that received volatile cues from genetically identical cuttings accumulated less natural damage than plants that received cues from non-self cuttings. Volatile communication is required to coordinate systemic processes such as induced resistance and plants respond more effectively to self than non-self cues. This self/non-self discrimination did not require physical contact and is a necessary first step towards possible kin recognition and kin selection.     

The social evolution of somatic fusion

The widespread potential for somatic fusion among different conspecific multicellular individuals suggests that such fusion is adaptive. However, because recognition of non-kin (allorecognition) usually leads to a rejection response, successful somatic fusion is limited to close kin. This is consistent with kin-selection theory, which predicts that the potential cost of fusion and the potential for somatic parasitism decrease with increasing relatedness. Paradoxically, however, Crozier found that, in the short term, positive-frequency-dependent selection eliminates the required genetic polymorphism at allorecognition loci. The 'Crozier paradox' may be solved if allorecognition is based on extrinsically balanced polymorphisms, for example at immune loci. Alternatively, the assumption of most models that self fusion is mutually beneficial is wrong. If fusion is on average harmful, selection will promote unconditional rejection. However, we propose that fusion within individuals is beneficial, selecting for the ability to fuse, but fusion between individuals on average costly, selecting for non-self recognition (rather than non-kin recognition). We discuss experimental data on fungi that are consistent with this hypothesis.     

Highly conserved,but highly specific: Somatic cell–cell fusion in filamentous fungi

The development of ascomycete fungal colonies involves cell–cell fusion at different growth stages. In the model fungus Neurospora crassa, communication of two fusing cells is mediated by an unusual signaling mechanism, in which the two partners take turns in signal sending and receiving. In recent years, the molecular basis of this unusual cellular behavior has started to unfold, indicating the presence of an excitable signaling network. New evidence suggests that this communication system is highly conserved in ascomycete fungi and, unexpectedly, even mediates interspecies interactions. At the same time, intricate allorecognition mechanisms were identified, which prevent the fusion of genetically unlike individuals. These observations suggest that signal specificity during fungal social behavior has not evolved on the level of signals and receptors, but is achieved at downstream checkpoints. Despite growing insight into the molecular mechanisms controlling self and non-self fungal interactions, their role in natural environments remains largely unknown.     

Autoreactive blood cells and programmed cell death in growth and development of protochordates

The tunicate Botryllus is a marine protochordate whose clonal colonies undergo regulated natural transplantations when they come into contact in nature. The outcome of these transplantations (fusion or rejection) is controlled by genes of a highly polymorphic histocompatibility system that resembles in many respects the mammalian major histocompatibility complex (MHC). While fusion or rejection reactions are often completed within 24 hr after transplantation, resorption of one partner of a pair of fused semiallogeneic colonies may occur days to weeks after initial contact. The latter process is similar to the degeneration of old individuals, or zooids, that precedes maturation of each new generation of asexual buds. Here we describe comparisons of in vitro reactions of a) mixtures of cells from allogeneic animals and b) cells taken from animals at the zooid-resorption ("takeover") stage of colony development. In vitro autoreactivity of cells from resorbing colonies may reflect in vivo responses to senescent cells, which in turn may be related to allorecognition events that govern fusion or rejection between colonies.     

The colonial urochordate Botryllus schlosseri: from stem cells and natural tissue transplantation to issues in evolutionary ecology

The urochordates, whose stem groups may have included the direct predecessors of the chordate line, serve as an excellent model group of organisms for a variety of scientific disciplines. One taxon, the botryllid ascidian, has emerged as the model system for studying allorecognition; this work has concentrated on the cosmopolitan species Botryllus schlosseri. Studies analyzing self-nonself recognition in this colonial marine organism point to three levels of allorecognition, each associated with different outcomes. The first level controls natural allogeneic rejections and fusions, in which blood-shared chimeras are formed. The second level leads to morphological resorption of partners within chimeras while the third allows the development of somatic and germ cell parasitic events. Recent studies on multi-chimeric entities formed in allogeneic fusions reveal evolutionary links between allorecognition, stem cell biology and ecology. Thus, the Botryllus system generates perspectives from different biological disciplines to yield a unique life history portrait.     

Self/non-self Discrimination in Basal Metazoa: Genetics of Allorecognition in the Hydroid Hydractinia

Colonial basal metazoans often encounter members of their ownspecies as they grow on hard substrata, with the encounterstypically resulting in either fusion of close relatives or rejectionbetween unrelated colonies. These allorecognition responsesplay a critical role in maintaining the genetic and physiologicalintegrity of the colony. Allorecognition responses in basalmetazoans are controlled by highly variable genetic systems.The molecular nature of such systems, however, remains to bedetermined. Current efforts to identify the genes and moleculescontrolling allorecognition in basal metazoans have followedtwo pathways: identification of molecules differentially expressedin incompatible interactions, and positional or map-based cloningof allorecognition genes. Most studies following the first approachhave been performed with marine demosponges, while those followingthe second approach have centered on the cnidarian of the genusHydractinia. Here, I discuss the latter, focusing primarilyon the genetic control of allorecognition responses.     

Self/nonself recognition in Cnidaria: contact to allogeneic tissue does not result in elimination of nonself cells in Hydra vulgaris

Although Cnidaria have no specialised immune cells, some colonial forms possess a genetic system to discriminate between self and nonself. Allorecognition is thought to protect them from fusion with genetically different individuals and to prevent germ line parasitism. Surprisingly, when grafting tissue of two species of the solitary freshwater polyp Hydra, we found within the contact zone phagocytozing epithelial cells which selectively eliminated cells from the other species (Bosch and David, 1986). This led us to speculate that Hydra, which never undergoes "natural transplantation", can differentiate between self and nonself (Bosch and David, 1986). In a previous paper (Kuznetsov et al., 2002) we described that cells which accumulate in the contact region of these interspecies grafts are apoptotic and that apoptosis is induced by impaired cell matrix contact. Thus, observations in such interspecies grafts did not give hints concerning the presence of a discriminative allorecognition system. To clarify whether this fundamental aspect of immunity is present in these phylogenetically old animals, we examined epithelial interactions between different strains of Hydra vulgaris. Here, we show that contact to allogeneic tissue does not evoke any response in terms of phagocytosis and elimination of allogeneic cells. We, therefore, question Hydra's ability to discriminate between self and nonself and propose that, in contrast to colonial cnidarians, the solitary polyp Hydra has either lost or substantially reduced this ability.     

Studies on Japanese botryllid ascidians. II. A new species of the genus Botryllus from the vicinity of Shimoda

Morphology and life history of a new species of the genus Botryllus belonging to the family Botryllidae were described in detail. This ascidian was collected from the stony shore in the vicinity of Shimoda (Shizuoka prefecture, Japan). The arrangement of ovary and testis in this ascidian was the same as that in other species of the genus Botryllus, while the embryo developed in a brood pouch formed from the invagination of peribranchial epithelium, as in the other genus Botrylloides. The processes and features of the allorecognition reaction of this ascidian were observed. The reaction showed the same processes as that in the species of the genus Botrylloides. Therefore, this ascidian has both features of the two genera of the family Botryllidae, which strongly suggests the necessity of reconsidering on the classificatory criteria of botryllid ascidians.     

Identification of vaccine candidate antigens of an ESBL producing Klebsiella pneumoniae clinical strain by immunoproteome analysis

Klebsiella pneumoniae is an opportunistic pathogen which causes pneumoniae, urinary tract infections and septicemia in immunocompromised patients. Hospital outbreaks of multidrug-resistant K. pneumoniae, especially those in neonatal wards, are often caused by strains producing the extended-spectrum-beta-lactamases (ESBLs). An immunoproteome based approach was developed to identify candidate antigens of K. pneumoniae for vaccine development. Sera from patients with acute K. pneumoniae infections (n = 55) and a control group of sera from healthy individuals (n = 15) were analyzed for reactivity by Western blot against ESBL K. pneumoniae outer membrane proteins separated by 2-DE. Twenty highly immunogenic protein spots were identified by immunoproteomic analysis. The immunogenic proteins that are most frequently recognized by positive K. pneumoniae sera were OmpA, OmpK36, FepA, OmpK17, OmpW, Colicin I receptor protein and three novel proteins. Two of the vaccine candidate genes, OmpA (Struve et al. Microbiology 2003, 149, 167-176) and FepA (Lai, Y. C. et al.. Infect Immun 2001, 69, 7140-7145), have recently been shown to be essential in colonization and infection in an in vivo mouse model. Hence, these two immunogenic proteins could serve as potential vaccine candidates.     

RNA based vaccines

The recognition that CD8(+) T-cell mediated Th1 immune responses were necessary to produce immunity to intracellular and transformed self pathogens led to intense interest in the delivery of nucleic acids, DNA, or RNA encoding candidate antigens, as vaccines. Antigen presenting cells (APC) encounter most protein and vaccine immunogens as extracellular proteins and, thus, present them on major histocompatibility complex (MHC) class II molecules leading to the activation of CD4(+) T cells. Protein antigens encoded by nucleic acids delivered to dendritic cell (DC) are produced inside the cell and, thus, can stimulate MHC class I mediated activation of CD8(+) T-cell immune responses. Unfortunately, DCs are not readily transfected with DNA (Akbari et al., 1999) resulting in the requirement for high concentrations of DNA and repeated immunizations to achieved immune responses. RNA, on the other hand, is readily taken up and expressed by DC, making it an alternative vaccine candidate. In this article, we will discuss immune responses developed, interactions between APC and RNA that activate and dictate DC activation, and preliminary studies using RNA in vivo and in vitro to develop protective immunity.