Allorecognition System Research Articles

Identification of immune-relevant genes in histoincompatible rejecting colonies of the tunicate Botryllus schlosseri

The colonial ascidian Botryllus schlosseri manifests a unique allorecognition system that is controlled by a single histocompatibility haplotype, the Fu/HC locus. When two allogeneic incompatible colonies come into direct contact, they develop inflammatory-like rejection lesions, called points of rejection (POR). While screening for differentially expressed genes during POR formation, we developed and analyzed a cDNA library of expressed sequence tags (ESTs) with 1693 unique ESTs that were clustered and assembled into 217 contigs and 1476 singlets. About 51% of these ESTs showed high similarity ( E-value ⩽0.005) to known database sequences, of which 123 matches were identified as immune-relevant genes encoding for stress proteins, pattern recognition receptors and complement proteins, proteases and protease inhibitors, cell adhesion and coagulation proteins, cytokine-related proteins, programmed cell death and proteasome-associated proteins. This first EST wide-screening analysis of the Botryllus allorecognition effector arm reveals a complex innate immune system, hallmarked by a whole genome response to allorecognition challenge.

How Botryllus Chooses to Fuse

In Botryllus schlosseri, a highly polymorphic allorecognition system limits the potential for vascular fusion by genetically dissimilar, adjacent colonies. In this issue, Nyholm et al. (2006) uncover the nature of the receptor that recognizes the products of the histocompatibility genes.

The importance of life-history stage and individual variation in the allorecognition system of a marine sponge

In marine invertebrates with complex life cycles, it may often be the case that trade-offs and behaviors differ between adult and larval stages. In this study, I examined the effects of life-history stage on allorecognition system function in the sponge, Haliclona sp. For sedentary marine invertebrates, allorecognition systems allow individuals to distinguish between genetically similar and distinct tissue they may encounter and are thought to reduce costly tissue fusion with individuals other than self or kin. Although it was found that sessile adults fused preferentially with self-tissue and exhibited a functioning allorecognition system, free-swimming larvae fused equally with sibling and non-sibling larvae resulting in swimming chimeras capable of successful metamorphosis, suggesting a stage-activated allorecognition system. In addition, adult sponges differed significantly in the propensity of their larvae to fuse suggesting variation in parental strategies. Analysis of larval swimming behavior indicated that larvae aggregate and are capable of increasing their encounters with other larvae and perhaps their probability of fusing in nature. The pursuit of fusion at this motile stage, along with evidence of a functioning adult allorecognition system, suggests that larvae may not express a recognition system, or that factors other than relatedness such as benefits to larval or adult chimeras, are involved in larval fusion and a stage-activated allorecognition system. In addition, this study demonstrates the presence of variation among individuals in the allorecognition system's ontogeny in the sponge Haliclona sp.

Genetic relationship and maturity state of the allorecognition system affect contact reactions in juvenile Seriatopora corals

Adult stony corals are characterized by a well-developed allorecognition system. In most cases, genetically identical coral grafts fuse, whereas genetically different grafts (allografts and xenografts) show various histoincompatible responses. Very little is known, however, about the allorecognition system in juvenile corals. We studied contact reactions of juvenile pairs in 2 sclerac- tinian coral species: Seriatopora caliendrum and S. hystrix (0 to 59 d after settlement). Juvenile colo- nies were experimentally grafted in combination groups of various genetic distances: sibling allo- grafts of S. hystrix (4 pairs), non sibling allografts of S. caliendrum (12 pairs), non sibling allografts of S. hystrix (16 pairs) and xenografts of the 2 species (26 pairs). Three tissue contact reactions were detected in all of the genetic combination groups: nonfusion, fusion and incompatible fusion. The transition in contact reaction patterns was recorded over a 1 yr period. Contact reactions and their duration were largely consistent among pair replicates of each combination and within each of the 4 genetic combination groups. We suggest that in juvenile Seriatopora corals, the genetic combination of each graft is a primary determinant of the contact reaction and its endurance. Maturity state of the allorecognition system, affects both onset and duration of the fusion and incompatible fusion reac- tions in the juvenile corals. Onset of the fusion reaction was observed only when grafted colonies were younger than 4 mo. Throughout the experimental period, most fusion reactions transformed into histoincompatible reactions or disconnection and virtually none of the allografts or xenografts main- tained stable chimeras. We suggest that as the alloimmune system matures, incompatible fusion reac- tions eventually transform into nonfusion or disconnection via tissue necrosis of the contact zone.

Role of a KIR/HLA-C allorecognition system in pregnancy*1

Role of a KIR/HLA-C allorecognition system in pregnancy*1

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.

Epithelial interactions in Hydra: apoptosis in interspecies grafts is induced by detachment from the extracellular matrix.

Apoptosis plays an important role in immunity and is widely used to eliminate foreign or infected cells. Cnidaria are the most basal eumetazoans and have no specialised immune cells, but some colonial cnidarians possess a genetic system to discriminate between self and non-self. By grafting epithelia of different species we have previously shown that the freshwater polyp Hydra eliminates non-self cells by phagocytosis. Here we have investigated whether apoptosis is involved in the histocompatibility reactions. We studied epithelial interactions between Hydra vulgaris and Hydra oligactis and show that a large number of apoptotic cells accumulate in the contact region of interspecies grafts. Histological analysis of the graft site revealed that displacement of the endodermal layer of Hydra vulgaris by endoderm from Hydra oligactis coincided with impaired cell-cell and cell-matrix contacts. We therefore suggest that in interspecies grafts, apoptosis is induced by the detachment of epithelial cells from the extracellular matrix (anoikis) and not by a discriminative allorecognition system.

The significance of women's natural killer cell receptors repertoire in the maintenance of pregnancy

It has been suggested that decidual NK cells play a regulatory role in pregnancy with their heterocladic (activating/inhibitory) receptor repertoire to control trophoblast infiltration. Most of the NK receptors (NKRs) have as ligands HLA molecules (HLA‐C,‐G,‐E), which are expressed on invading trophoblast and they may interact with their NKR counterparts and provide self‐signals to evade trophoblastic damage.When we studied the NKR repertoire in couples with unexplained recurrent spontaneous abortions by genotyping partners for NKRs (KIR and CD94/NKG receptors, known to have as ligands HLA molecules present on trophoblast), we found that aborting women usually have a limited inhibitory KIR (inhKIR) repertoire.Our hypothesis that a limited inhKIR repertoire predisposes to miscarriage, because it is ineffective to abort activating signals and protect trophoblast, is supported by studies in selected couples, known to posses HLA‐Cw antigens interacting with inhKIRs and by studies on the placental material of spontaneously lost or electively terminated pregnancies. Most of the aborters did not have inhKIRs specific for the HLA‐C antigens possibly expressed on trophoblast and this seems to be confirmed when KIRs and HLA‐Cs are genotyped using DNA extracted from decidual and trophoblastic cells, respectively.Increasing knowledge about KIR haplotypes is expected to be helpful in the study of the suggestedinhKIR – HLA‐C allorecognition system in pregnancy.

The ontogeny of allorecognition in a colonial hydroid and the fate of early established chimeras.

Colonies of the marine hydroid, Hydractinia, are able to discriminate between their own tissues and those belonging to unrelated conspecifics. We have studied the ontogeny of this allorecognition system by a series of allogeneic transplantations along a developmental gradient, including two-cell-stage embryos, 8 h morulae, planula larvae and metamorphosed polyps. Allograft acceptance of incompatible tissue was observed in all embryonic and larval stages, whereas metamorphosed polyps rejected incompatible transplanted allografts. Most of the chimeras established at the two-cell-stage, although composed of two allogeneic, incompatible entities with mismatching allorecognition loci, developed normally and remained stable through metamorphosis. The results of post metamorphic transplantation assays among the chimeras and the naive ramets, suggested that both incompatible genotypes were still represented in the chimera despite the onset of alloimmune maturation. The naive colonies always rejected each other. Chimeras established from later embryonic and larval stages did not develop into adult chimeric entities, but rather separated immediately post metamorphosis. We thus show that (1) allorecognition in this species matures during metamorphosis and (2) genetically incompatible entities may coexist in one immunologically mature, chimeric soma, provided that they were grafted early enough in ontogeny.

The hydroid Hydractinia: a versatile, informative cnidarian representative.

The Cnidaria represent the most ancient eumetazoan phylum. Members of this group possess typical animal cells and tissues such as sensory cells, nerve cells, muscle cells and epithelia. Due to their unique phylogenetic position, cnidarians have traditionally been used as a reference group in various comparative studies. We propose the colonial marine hydroid, Hydractinia, as a convenient, versatile platform for basic and applied research in developmental biology, reproduction, immunology, environmental studies and more. In addition to being a typical cnidarian representative, Hydractinia offers many practical and theoretical advantages: studies that are feasible in Hydra like regeneration, pattern regulation, and cell renewal from stem cells, can be supplemented by genetic analyses and classical embryology in Hydractinia. Metamorphosis of the planula larva of Hydractinia can be used as a model for cell activation and communication and the presence of a genetically controlled allorecognition system makes it a suitable model for comparative immunology. Most importantly, Hydractinia may be manipulated at most aspects of its (short) life cycle. It has already been the subject of many studies in various disciplines, some of which are discussed in this essay.

Behavior of Hemocytes in the Allorejection Reaction in Two Compound Ascidians, Botryllus scalaris and Symplegma reptans.

In botryllid ascidians, the type of allorejection reaction differs among species. Comparative studies of these different reactions contribute to our understanding of the allorecognition and nonself-rejection system. We studied the morphology of hemocyte behavior during allorejection reactions in two species, Botryllus scalaris and Symplegma reptans, which stand at important points in botryllid phylogeny. In B. scalaris, phagocytes mediated hemocyte aggregation, resulting in interruption of blood flow just after vascular fusion of incompatible colonies. Although previous studies indicate that morula cells (MCs) play a central role in the rejection reaction, the MCs of B. scalaris did not participate in the rejection reaction. Colonies of S. reptans showed two types of allorejection reaction that started at different points in the process of vascular fusion between two colonies. In both types of rejection reaction, the MCs played a central role and behaved similarly to those of all botryllids except B. scalaris and a botryllid from Israel. These observations suggest that the differences in hemocyte behavior and allorecognition site observed in this study reflect the variation in allorejection reactions among botryllids.

KIN INTERACTIONS IN A COLONIAL HYDROZOAN (HYDRACTINIA SYMBIOLONGICARPUS): POPULATION STRUCTURE ON A MOBILE LANDSCAPE.

Many sessile colonial organisms intensively compete with conspecifics for growing space. This competition can result in either cooperative fusion or aggressive rejection between colonies, and some species have evolved highly polymorphic genetic systems that mediate the outcome of these interactions. Here we demonstrate the potential for interactions among close kin as the basis for the evolutionary maintenance of a genetically polymorphic allorecognition system in the colonial hydroid Hydractinia symbiolongicarpus, which lives on gastropod shells occupied by hermit crabs. Fusion between hydroids in the laboratory is restricted mainly to encounters between full siblings, whereas other encounters result in aggressive rejection. Natural selection acting on the costs or benefits of fusion between colonies could be responsible for the present maintenance of such a highly specific behavioral response, but only if encounters between fusible colonies still occur in contemporary populations. The large size of these hydroid populations and the mobility of the crabs should limit the potential for interactions among closely related hydroids on the same shell. However, RAPD polymorphisms among a large sample of hydroids from a population off the coast of Massachusetts indicate that genetically similar colonies are often found together on the same shell. Some genetic distances between colonies on the same shell were low relative to genetic distances between colonies on different shells or genetic distances between known full siblings from laboratory matings. We conservatively estimate that 2-18% of co-occurring colonies may be full sibling pairs. These observations suggest that encounters between genetically similar hydroids are common, despite the mobile nature of their habitat, and these encounters may provide frequent opportunities for natural selection to influence the evolution of cooperative and agonistic behaviors and their polymorphic genetic basis.

Not like Botryllus: indiscriminate post–metamorphic fusion in a compound ascidian

Fusion trials between metamorphs of the aplousobranch compound ascidian Diplosoma listerianum indicated that chimera formation was not dependent on relatedness. Similar, high rates of union were observed between full siblings, half siblings, unrelated individuals from the same population, and individuals from two geographically distant localities. This is in contrast to the well–studied ascidian genus Botryllus , in which a highly polymorphic allorecognition system governing the fusion–non–fusion reaction (colony specificity) largely limits fusion to close relatives. Fusion in Botryllus establishes a vascular chimera throughout which stem cells may circulate, promoting cell lineage competition between the fusion partners. The restriction of fusion to close kin in Botryllus is thought to reduce the inclusive fitness costs of competitive interactions between cell lineages within the chimera. In contrast to Botryllus , modules (zooids) of a D. listerianum colony are not interlinked by blood vessels, seemingly precluding the exchange of stem cells. The apparent absence of strict colony specificity in D. listerianum is thus in keeping with the predictions of the Botryllus model for the maintenance of allorecognition polymorphism. However, colony specificity has been reported in other species of aplousobranch ascidian that also lack a common vascular system. In these, the threat of migrating blood–borne stem cells cannot be responsible for the presence of colony specificity. One possibility, requiring experimental investigation, is that stem cells could perhaps migrate between zooids by another route, such as through the matrix of the colonial tunic. Even in the absence of stem cell exchange, cheating on the costs of colony maintenance and defence could also produce selective forces favouring colony specificity. In compound ascidians, this could involve unequal contribution to extrazooidal structures, principally the tunic and related tissues. This consideration seems potentially relevant to the lack of discrimination during fusion in D. listerianum , since extrazooidal somatic investment in this species appears minimal, severely limiting the scope for this other form of cheating. The various possible modes of exploitative interaction between fused colonies are not mutually exclusive, and offer fundamentally similar explanations for colony specificity. If none of them can be shown to occur in non–botryllid species possessing colony specificity, the generality of the Botryllus model may require re–evaluation.

Transplantation of Fu/HC-incompatible zooids in Botryllus schlosseri results in chimerism.

The colonial urochordate Botryllus schlosseri undergoes a genetically defined, natural transplantation reaction that is controlled by a single Mendelian locus (called the Fu/HC). This Fu/HC-based allorecognition system is initiated when peripheral elements of the vasculature interact on the edges of two asexually expanding colonies. To better understand the spatial organization of the cellular elements responsible for Fu/HC-based allorecognition, we bypassed the normal site of interaction (the ampullae) and experimentally transplanted zooids between Fu/HC-noncompatible Botryllus schlosseri pairs. The results show that (1) instead of the expected rejections (tissue necroses) that develop after natural contacts between peripheral blood vessels, the transplanted organs are morphologically eliminated within a few days in conjunction with the normal blastogenic cycle; and (2) donor-recipient chimerism is established after complete morphological elimination of transplanted tissues. These results suggest that Fu/HC-based allorecognition responses in Botryllus schlosseri occur exclusively at the ampullae and that once cells have crossed this barrier, they are able to survive and proliferate in the new host colony.

EVOLUTION OF ALLORECOGNITION IN BOTRYLLID ASCIDIANS INFERRED FROM A MOLECULAR PHYLOGENY.

Despite the functional and phyletic ubiquity of highly polymorphic genetic recognition systems, the evolution and maintenance of these remarkable loci remain an empirical and theoretical puzzle. Many clonal invertebrates use polymorphic genetic recognition systems to discriminate kin from unrelated individuals during behavioral interactions that mediate competition for space. Space competition may have been a selective force promoting the evolution of highly polymorphic recognition systems, or preexisting polymorphic loci may have been coopted for the purpose of mediating space competition. Ascidian species in the family Botryllidae have an allorecognition system in which fusion or rejection between neighboring colonies is controlled by allele-sharing at a single, highly polymorphic locus. The behavioral sequence involved in allorecognition varies in a species-specific fashion with some species requiring extensive intercolony tissue integration prior to the allorecognition response, while other species contact opposing colonies at only a few points on the outer surface before resolving space conflicts. Due to an apparent species-specific continuum of behavioral variation in the degree of intercolony tissue integration required for allorecognition, this system lends itself to a phylogenetic analysis of the evolution of an allorecognition system. We constructed a molecular phylogeny of the botryllids based on 18S rDNA sequence and mapped allorecognition behavioral variation onto the phylogeny. Our phylogeny shows the basal allorecognition condition for the group is the most internal form of the recognition reaction. More derived species show progressively more external allorecognition responses, and in some cases loss of some features of internal function. We suggest that external allorecognition appears to be a secondary function of a polymorphic discriminatory system that was already in place due to other selective pressures such as gamete, pathogen, or developmental cell lineage recognition.

Linkage analysis of HSP70 genes and historecognition locus in botryllus schlosseri.

The protochordate allorecognition system has long invited comparison with the vertebrate major histocompatibility complex (MHC). In the colonial species Botryllus schlosseri, a rapid fusion or rejection response resembling graft acceptance or rejection in vertebrates is controlled by a single highly polymorphic genetic region. Because linkage between heat shock protein 70 (HSP70) genes and the MHC appears to be conserved within the vertebrate lineage, linkage relationships between two HSP70 genes (HSP70.1 and HSP70.2) and the historecognition locus (FuHC) have been analyzed in B. schlosseri. Segregation patterns of restriction fragment length polymorphisms located in the 3' flanking regions of HSP70.1 and HSP70.2 were determined for progeny of defined crosses. These progeny were also analyzed for fusibility type by an in vivo cut colony assay. No close linkage was detected between any of the three loci. These results do not support the hypothesis that the allorecognition response in B. schlosseri is determined by an MHC homologue. However, it remains a possibility that orthologues of other MHC-linked genes will be linked to the B. schlosseri FuHC.

Differential regulation of T cell activation for primary versus secondary proliferative responses.

T cell activation in vivo results in proliferation and generation of effector cytokine-secreting cells, as also in development of memory cells that mount enhanced responses upon restimulation. However, differences in the signals promoting generation of effector vs memory T cells are not yet characterized. In this study, using various strategies to modulate an allorecognition system for priming human T cells in vitro, we show that there are indeed differences between the signaling requirements for a first proliferative response and those for priming T cells for enhanced recall proliferative responses. Using APCs fixed with varying concentrations of paraformaldehyde, we show that the loss of ability of these APCs to generate a first response is not matched by a similar loss in their ability to prime responder T cells for recall responses. Prevention of DNA replication during T cell priming with aphidicolin, a DNA polymerase inhibitor, is not inimical to successful T cell priming. Thus, clonal expansion during priming is less crucial than the primed activation status of T cells for the enhanced recall response. We also show that pentoxifylline, a phosphodiesterase inhibitor, inhibits the primary proliferative response, but its presence during priming enhances the recall response capabilities of T cells. On the other hand, the presence of the calcineurin inhibitor cyclosporin A during priming reduces the efficiency of priming, but at low concentrations it induces, like pentoxifylline, enhancement in recall response capability. These findings have significant implications in designing immunosuppressive therapy and in the analysis of signals for T cell memory commitment.

Human uterine NK cells have a similar repertoire of killer inhibitory and activatory receptors to those found in blood, as demonstrated by RT-PCR and sequencing

The expression of natural killer (NK) cell receptors specific for HLA class I molecules has been studied in CD56 bright, CD3 − NK cells isolated from the pregnant uterine mucosa, the decidua. RT-PCR was performed on cDNA from uterine NK cells with primers designed to amplify members of the killer inhibitory receptor (KIR)/killer activatory receptor (KAR) gene family. Sequencing of the PCR products revealed that uterine NK cells express KIR/KAR which have two or three extracellular immunoglobulin superfamily (Ig-SF) domains. NK receptors for both groups of HLA-C alleles were found. KIR, characterised by a long cytoplasmic tail containing the immune receptor tyrosine-based inhibitory motif (ITIM), and KAR, characterised by a short cytoplasmic domain with a transmembrane region containing a charged lysine, were both identified. Different individuals appear to have a distinct but overlapping repertoire of KIR/KAR. No new members of this NK receptor gene family were identified in the uterine CD56 bright NK cells. Similar findings were obtained from non-pregnant endometrial tissues representative of different stages of the menstrual cycle. Immunohistology confirmed that the KIR protein products were expressed by decidual NK cells. These results reveal that NK receptors for trophoblast HLA class I molecules are present in maternal uterine NK cells. Fetal trophoblast cells infiltrating the decidua express HLA-G and HLA-C gene products. This suggests that maternal recognition of the fetus may be mediated by an NK allo-recognition system.

Somatic and germ cell parasitism in a colonial ascidian: Possible role for a highly polymorphic allorecognition system

A colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction in the wild that results alternatively in colony fusion (chimera formation) or inflammatory rejection. A single, highly polymorphic histocompatibility locus (called Fu/HC) is responsible for rejection versus fusion. Gonads are seeded and gametogenesis can occur in colonies well after fusion, and involves circulating germ-line progenitors. Buss proposed that colonial organisms might develop self/non-self histocompatibility systems to limit the possibility of interindividual germ cell “parasitism” (GCP) to histocompatible kin [Buss, L. W. (1982) Proc. Natl. Acad. Sci. USA 79, 5337–5341 and Buss, L. W. (1987) The Evolution of Individuality (Princeton Univ. Press, Princeton]. Here we demonstrate in laboratory and field experiments that both somatic cell and (more importantly) germ-line parasitism are a common occurrence in fused chimeras. These experiments support the tenet in Buss’s hypothesis that germ cell and somatic cell parasitism can occur in fused chimeras and that a somatic appearance may mask the winner of a gametic war. They also provide an interesting challenge to develop formulas that describe the inheritance of competing germ lines rather than competing individuals. The fact that fused B. schlosseri have higher rates of GCP than unfused colonies additionally provides a rational explanation for the generation and maintenance of a high degree of Fu/HC polymorphism, largely limiting GCP to sibling offspring.

Somatic and germ cell parasitism in a colonial ascidian: possible role for a highly polymorphic allorecognition system.

A colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction in the wild that results alternatively in colony fusion (chimera formation) or inflammatory rejection. A single, highly polymorphic histocompatibility locus (called Fu/HC) is responsible for rejection versus fusion. Gonads are seeded and gametogenesis can occur in colonies well after fusion, and involves circulating germ-line progenitors. Buss proposed that colonial organisms might develop self/non-self histocompatibility systems to limit the possibility of interindividual germ cell "parasitism" (GCP) to histocompatible kin [Buss, L. W. (1982) Proc. Natl. Acad. Sci. USA 79, 5337-5341 and Buss, L. W. (1987) The Evolution of Individuality (Princeton Univ. Press, Princeton]. Here we demonstrate in laboratory and field experiments that both somatic cell and (more importantly) germ-line parasitism are a common occurrence in fused chimeras. These experiments support the tenet in Buss's hypothesis that germ cell and somatic cell parasitism can occur in fused chimeras and that a somatic appearance may mask the winner of a gametic war. They also provide an interesting challenge to develop formulas that describe the inheritance of competing germ lines rather than competing individuals. The fact that fused B. schlosseri have higher rates of GCP than unfused colonies additionally provides a rational explanation for the generation and maintenance of a high degree of Fu/HC polymorphism, largely limiting GCP to sibling offspring.