Colonial allorecognition,hemolytic rejection,and viviparity in botryllid ascidians

Allorecognition is a fundamental system that animals use to maintain individuality. Although embryos are usually semiallogeneic with their mother, viviparous animals are required to allow these embryos to develop inside the maternal body, but must also eliminate an "invasion" by nonself. In colonial ascidians of the family Botryllidae, when two colonies are brought into contact at their growing edges, a hemolytic rejection reaction occurs between allogeneic colonies. Morula cells, a type of hemocyte, are the major effector cells in the hemolytic rejection. Morula cells infiltrate and aggregate where the two colonies make contact, and then discharge their vacuolar contents, which contain phenoloxidase and quinones. In viviparous botryllids, colonial contact at artificially cut surfaces always results in colonial fusion and establishment of a common vascular network even between allogeneic colonies in which the growing-edge contact results in rejection. This colonial fusion between incompatible colonies (surgical fusion) suggests that the allorecognition sites are not distributed in the vascular system in which the embryos are brooded. It is supposed that a common ancestor of the viviparous species lost the capacity for allorecognition in their vascular system to protect its embryos from alloreactivity, when it changed from ovoviviparous to viviparous in the course of evolution. The limited distribution of allorecognition sites would be a solution to the embryo-parent histoincompatibility in viviparity.     

The grapes of incompatibility

When two colonies of the tunicate Botryllus contact each other, they either fuse into a single colony or mount a destructive reaction, which keeps them apart. Which of the two reactions takes place is determined by a single, highly polymorphic fusion/histocompatibility (FuHC) locus. Recent communications report the cloning and characterization of the FuHC locus and suggest that its function may be to protect against parasitism by conspecific stem cells.     

Colony specificity in the xenogeneic combinations among four Botrylloides species (urochordata,ascidiacea)

Xenogeneic rejection reactions were histologically examined among four compound ascidians of the genus Botrylloides; B. simodensis, B. lentus, B. fuscus and B. violaceus, to compare with the allogeneic rejections of these species. When the incompatible conspecifics were brought into contact, hemolytic rejections occurred at the point where the tunic of the two colonies was partially fused. Xenogeneic contact at their growing edges induced hemolytic rejection in some combinations (B. simodensis-B. lentus, B. lentus-B. fuscus, and B. fuscus-B. violaceus), while conspicuous reaction was not found in the other combinations. Since the hemolytic rejection requires the partial fusion of tunic, the occurrence of hemolytic rejection suggests that the tunic cuticle of the colonies does not discriminate the facing colony from conspecifics. On the other hand, whereas cut surface contact between incompatible conspecifics induced intense rejection in B. simodensis, it resulted in fusion (formation of vascular connection) even in the combination in which the growing edge contact resulted in rejection. In xenogeneic combination, the cut surface contact of colonies always resulted in an intense rejection reaction except for B. fuscus-B. violaceus in which hemolytic reactions did not occur. The absence of hemolytic rejection suggests that the effector system for rejection reaction is not activated in this combination. Activity of phenoloxidase, a key enzyme of the rejection reaction, indicates lower reactivity in B. lentus, B. fuscus and B. violaceus than that in B. simodensis.     

A complex allorecognition system in a reef-building coral: delayed responses,reversals and nontransitive hierarchies

A highly polymorphic and complex allorecognition system in the coral Stylophora pistillata was revealed in the field by assaying branch pair combinations among 11 colonies (181 assays) for 24 months. Replicates of between-colony combinations exhibited consistent outcomes, in both time scale and type of response. Different allogeneic combinations exhibited one of two main outcomes, either unilateral rejection, or an array of other incompatible reactions following a state of non-fusion. These responses were partially linked with color morphs (purple dominated yellow). An additional 22 isogeneic grafts resulted in complete fusion. Unilateral rejection occurred 1–7 months following initial contact. Nonfusion usually developed into skeletal suture barriers after 3–9 months, and then into unilateral colony-specific overgrowths at 6–23 months with some reversals in direction at 18–22 months. During this process, small lesions usually developed on the tissue of the subordinate partner, which were either overgrown by the dominant partner or healed. After two years, a network of overgrowths among colonies was established with essentially hierarchial properties, but some nontransitive interactions also occurred. The colonies segregated into three distinct histocompatibility groups; within each group, colonies engaged in nonfusion. Between groups, colonies exhibited nonfusion or rejected each other in a group-specific manner. Based on the results, we discuss the terminology used for fusion versus rejection phenomena in scleractinian corals, the possible genetic background for self-nonself recognition in Stylophora, and the methodological artifacts associated with the use of short-term allorecognition assays. Correspondence to: B. Rinkevich     

Colony Specificity in the Colonial Tunicate Botryllus and the Origins of Vertebrate Immunity

SYNOPSIS. Colonies of the compound tunicate Botryllus show thecapacity for self—nonself discrimination by fusion betweenseparated pieces of the same colony and rejection between piecesof unrelated colonies. We have found that genes controllingthis colony specificity are similar to those which cause transplantrejection in the vertebrates. Like the loci within the vertebratemajor histocompatibility complex (MHC), Botryllus fusibility(or histocompatibility) genes are highly polymorphic. In Botryllus,the histocompatibility complex also controls self—sterility,and limits cross—fertilization between colonies sharinghistocompatibility alleles. The mouse MHC, the H-2 region, islinked to loci which also affect the frequencies of allelesat H-2 loci in mouse populations. Thus both systems containcharacters which could act to promote the heterozygous conditionat the linked histocompatibility loci. We suggest that suchlinked characters are responsible for the evolution of allogeneicpolymorphism in vertebrates (however currently maintained),and that tunicate fusibility loci may be the evolutionary precursorsof vertebrate MHC genes.     

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.     

Genotypic variability following fusion in the invasive colonial tunicate Didemnum vexillum

Fusion to form a chimera has been documented in many marine invertebrate taxa, including poriferans, cnidarians, bryozoans, and colonial ascidians. Allogenic interactions in chimeric ascidian colonies vary widely across taxonomic groups but are poorly characterized in the invasive colonial ascidian Didemnum vexillum. The moderate level of discrimination expressed in the fusion–rejection response of D. vexillum suggests that there is some integration of cells beyond the fusion line in a chimeric colony. We tracked the shifts in representation of microsatellite alleles between fused colonies of D. vexillum to elucidate the extent of genotypic integration in fused colonies and the patterns of changes to the genotypic composition of colonies immediately following chimera formation. By genotyping colonies before and after fusion, we found that allogeneic fusion in D. vexillum may lead to genotypic changes beyond the visible fusion line. Alleles from one colony were found in multiple tissue samples in the chimera 7–10 days after fusion had occurred. In some instances, alleles that were in a single colony prior to fusion were lost following fusion. We observed multiple patterns of allelic change, including both the unidirectional transfer and reciprocal exchange of alleles between fused colonies. Our findings suggest that tissue or cells are exchanged following allogeneic fusion between colonies of D. vexillum and that the genotypic composition of chimeric colonies may be fluid.     

Tissue compatibility between colonies and between newly settled larvae of Pocillopora damicornis

Grafting experiments with newly settled larvae and with adult colonies of Pocillopora damicornis were performed. When pairs of newly settled larvae released from different colonies were kept in contact, they fused to form an aggregated colony. Even newly settled larvae derived from colonies belonging to different color morphs fused with each other and no sign of allogeneic rejection was observed. However, when branches of adult colonies belonging to different color morphs were kept in contact, they did not fuse. Fusion was observed only when branches derived from the same colony were paired. The present results suggest that juvenile corals lack the functional histocompatibility system as shown by adult colonies.     

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     

MHC alloantigens elicit secondary,but not primary,indirect in vitro proliferative responses

The relative contributions of direct and indirect pathways of allorecognition to graft rejection remain controversial. Recent reports suggest that the indirect pathway may play a prominent role in both acute and chronic allograft rejection. Such studies suggest that MHC-derived allopeptides are more immunogenic than those derived from minor histocompatibility or other nominal Ags. The aim of this study was to characterize the immunogenicity of MHC alloantigens in MHC-defined miniature swine via primary and secondary MLR culture assays. APCs were selectively depleted from either responder or stimulator cell populations to specifically analyze direct and indirect proliferative responses, respectively. Radio-resistant cytokine secretion and subsequent backstimulation of responder cells was eliminated by using stimulators that were either lysed or unresponsive to the responder MHC haplotypes. When the effect of backstimulation was eliminated from MLR culture assays, indirect proliferative responses were not observed among naive responders. Only after in vivo priming of responder animals could indirect proliferation be detected. These data do not refute the potential importance of indirect allorecognition in graft rejection. However, they suggest that MHC-derived alloantigens behave similarly in vitro to minor histocompatibility Ags, with comparable immunogenicity. These data also suggest that the MLR culture assay does not accurately reflect the importance of indirect mechanisms that have previously been reported in experimental models of graft rejection. A greater understanding of the indirect pathway and the associated immunogenicity of MHC allopeptides has the potential benefit of enabling the development of therapeutic interventions to prevent or halt allograft rejection.     

Allogeneic interactions in Hydractinia: is the transitory chimera beneficial?

The colonial marine hydroid, Hydractinia, exhibits four possible outcomes to allogeneic contacts: passive rejection, aggressive rejection, stable fusion and transitory fusion. In the special case of transitory fusion, Hydractinia colonies undergo tissue fusion, followed by tissue death at the original contact area, and colony separation. This type of rejection is different in several aspects from the rejection process that accompanies incompatible encounters. It has been suggested that in transitory fusion, the colonies gain immediate benefits from fusion, mainly due to size increase, without succumbing to costs associated with fusion (germ line parasitism). We report a long-term observation of repeated fusion and separation cycles in clones featuring transitory fusion that revealed a slow-down of specific growth rates following fusion, and recovery in growth rates following separation. Very rapid transfer of stained material between partners in transitory chimeras provides suggestive evidence that protection against germ line parasitism is far from being guaranteed by separation. Our data cast doubt as to whether the benefits considered for transitory fusion are sustainable and support the already made suggestion that fusion with self, rather than fusion with kin, has been the major selective force governing the evolution of allorecognition in colonial invertebrates.     

Xenogeneic rejection among three botryllids (compound Ascidians)

Xenogeneic rejection was observed among colonies of three botryllids, Botryllus scalaris, Botryllus primigenus, and Botrylloides simodensis. Allogeneic recognition occurs in each of these species, but the manner of allogeneic rejection differs among them. We studied xenogeneic rejection reactions among these species under the following conditions: colony contact at natural growing edges, colony contact at artificially cut surfaces, and injection of xenogeneic blood plasma into a vascular vessel. In the first two cases, xenogeneic rejection occurred only in Botryllus primigenus and Botrylloides simodensis. The features of that xenogeneic rejection were similar to those of allogeneic rejection in each of these two botryllids. Injection of xenogeneic blood plasma induced responses similar to those of allogeneic rejection in all three botryllids. It is interesting to note that colonies of Botryllus scalaris never showed any response against injected blood plasma from allogeneic incompatible colonies, unlike the responses seen in colonies of the other two botryllids under the same conditions. On the basis of these results, the relationship between allogeneic and xenogeneic rejection in botryllids is discussed.     

Stem cells are units of natural selection in a colonial ascidian

Stem cells are highly conserved biological units of development and regeneration. Here we formally demonstrate that stem cell lineages are also legitimate units of natural selection. In a colonial ascidian, Botryllus schlosseri, vascular fusion between genetically distinct individuals results in cellular parasitism of somatic tissues, gametes, or both. We show that genetic hierarchies of somatic and gametic parasitism following fusion can be replicated by transplanting cells between colonies. We prospectively isolate a population of multipotent, self-renewing stem cells that retain their competitive phenotype upon transplantation. Their single-cell contribution to either somatic or germline fates, but not to both, is consistent with separate lineages of somatic and germline stem cells or pluripotent stem cells that differentiate according to the niche in which they land. Since fusion is restricted to individuals that share a fusion/histocompatibility allele, these data suggest that histocompatibility genes in Botryllus evolved to protect the body from parasitic stem cells usurping asexual or sexual inheritance.     

SELF-RENEWAL OF COLONY FORMING UNITS (CFU) IN SERIAL BONE MARROW TRANSPLANTATION EXPERIMENTS

The capacity of stem cells (CFU) for self-renewal was tested by transplanting normal bone marrow (primary transplantation) and bone marrow which had been subjected to one or two earlier transplantations (secondary and tertiary transplantation) into lethally irradiated syngeneic recipients. It was found that the capacity for self-renewal is diminished within the first weeks after one or more previous transplantations. This ability of stem cells recovered after a longer interval after the previous transplantation. The time required for this recovery depended upon the number of previous transplantations and amounted to more than 1 or 2 months after one or two transplantations respectively. Shortly after transplantation the CFU/nucleated cell ratio in bone marrow was below normal and its decrease was more pronounced when the bone marrow had been transplanted more often. An increase of the ratio towards normal values was observed in the course of one month after the last transplantation. Measurements of the spleen colony size after transplantation of normal and re-transplanted bone marrow indicated that CFUs from re-transplanted marrow gave slightly smaller spleen colonies than those of normal marrow.
It is concluded that the decreased self-renewal of stem cells shortly after previous transplantations is probably not due to a limitation in the number of normal mitoses they can perform, but to a loss of stem cells by transfer to the compartment of differentiating cells.     

Prevention of early rejection of neural xenotransplants in the rat brain by the influence of magnetic field

The effect of alternating low-frequency (50 Hz, 40 mT) magnetic field (MF) on preventing of early rejection of xenotransplants (XT) of the chicken embryo forebrain, grafted in the brain parenchyma of adults rats, was studied. For this purpose, rats with XT were treated with 1-h-long MF applications over 1, 3, and 5 days following neurotransplantation. The animals with XT, but without treatment with MF, were used as a control. Morphological analysis of XT and neighboring brain tissues of recipients was performed 5 days after transplantation. It was found that the action of MF prevented or substantially weakened reactions of XT rejection at early stages after XT grafting in the brain of recipient rats. Destruction of the neighboring brain tissues was decelerated, while in the control group of animals destructions were clearly manifested. Positive effect of MF was observed even after single 1-h-long application of MF the next day after the operation, and it did not change when MF treatment was repeated 3 or 5 times during the following days. It is suggested that MF depresses some cellular reactions, in particular migration of lymphocytes and reactive gliosis, which cause early XT rejection. A possibility that the MF effect is due to activation of immunodepressant factor and/or to blockade of antigen receptors of the main histocompatibility complex on the donor and/or recipient cell surface cannot be ruled out.     

Relationships among hemocytes, tunic cells, germ cells, and accessory cells in the colonial ascidian Botryllus schlosseri

Monoclonal antibodies were raised against hemocytes of the colonial ascidian Botryllus schlosseri as possible tools to study hemocyte differentiation. In this species, blood cells are involved in various biological functions, such as immunosurveillance, encapsulation of foreign bodies, metal accumulation, and allorecognition. The latter process drives the fusion or rejection of contacting colonies, according to whether they do or do not share at least one allele at the fusibility/histocompatibility (Fu/HC) locus. Hemocytes take part in the rejection reaction, which suggests that they express molecules, coded by the Fu/HC locus, on their surface. A homozygous colony at the Fu/HC locus was used to produce the antibodies, which were screened by immunocytochemistry on hemocyte monolayers, immunohistochemistry on colony paraffin sections, and immunoblotting on colony homogenates. Here, we report on one of the obtained antibodies (1D8), which recognized a surface epitope on hemocytes of the donor colony and other colonies, apparently in a manner specific to the Fu/HC genotype. It also labeled a single 80-kDa band in colony homogenates. In addition, it specifically recognized tunic cells, germ cells, and their accessory cells. These results strengthen the assumption of a close relationship among these types of cells and blood cells, and suggest a close relationship among the above cells, probably deriving from undifferentiated blood cells.     

Allogeneic inhibition in a compound ascidian, Batryllus primigenus Oka. I. Processes and features of "Nonfusion" reaction

The processes and features of the “Nonfusion” reaction (NFR), a measure positive allogeneic inhibition, in a compound ascidian, Botryllus primigenus, has been studied.The process and the features of NFR have been observed when two incompatible colonies were placed as to make a contact with (1) ampullae to ampullae, (2) ampullae to margin without ampullae, and (3) cut surface to cut surface of the colonies. NFR appeared immediately after fusion of the test matrices of two incompatible colonies. The ampullae have the ability to loosen or dissolve the outer membrane of the test matrix and to penetrate into the other colony. The first response in NFR was the destruction of test cells around the contact area leading to an appearance of filaments around disintegrated test cells. The second reaction was contraction of the ampullae. Distal parts of the ampullae were thus cut off by constriction and necrosis. The contact area between two incompatible colonies was then separated from the healthy parts of both colonies, and NFR was completed.     

Contact of human immunodeficiency virus type 1-infected and uninfected CD4+ T lymphocytes is highly cytolytic for both cells.

Individuals infected with the human immunodeficiency virus (HIV) experience a marked loss of CD4+ T lymphocytes, leading to fatal immunodeficiency. The mechanisms causing the depletion of these cells are not yet understood. In this study, we observed that CD4+ T lymphocytes from HIV type 1 (HIV-1)-infected and uninfected individuals rapidly lysed B lymphoblasts expressing the HIV-1 envelope glycoprotein on the cell surface and Jurkat cells expressing the complete virus. Contact of uninfected CD4+ T cells with envelope glycoprotein-expressing cells also resulted in the lysis of the uninfected CD4+ T cells. Cytolysis did not require priming or in vitro stimulation of the CD4+ T cells and was not restricted by major histocompatibility complex molecules. Cytotoxicity was inhibited by soluble CD4 and anti-CD4 monoclonal antibodies that block binding of CD4 to gp120. In addition, neutralizing anti-CD4 and anti-gp120 monoclonal antibodies which block postbinding membrane fusion events and syncytium formation also inhibited cell lysis, suggesting that identical mechanisms in HIV-infected cultures underlie cell-cell fusion and the cytolysis observed. However, cytotoxicity was not always accompanied by the formation of visible syncytia. Rapid cell lysis after contact of uninfected and HIV-1-infected CD4+ T cells may explain CD4+ T-cell depletion in the absence of detectable syncytia in infected individuals. Moreover, because of its vigor, lysis of envelope-expressing targets by contact with unprimed CD4+ T lymphocytes may at first glance resemble antigen-specific immune responses and should be excluded when cytotoxic T-lymphocyte responses in infected individuals and vaccinees are evaluated.     

Analysis of the major histocompatibility complex in Syrian hamsters. III. Cellular and humoral immunity to alloantigens.

Among the genetic loci incorporated into the major histocompatibility complex in every species studied to date have been prominent genes encoding for strong histocompatibility determinants that elicit detectable alloantibody responses and which are the chief antigenic targets of cell-mediated cytotoxicity reactions. The K and D regions of the H-2 complex in the mouse and the A, B, and C regions of the HLA complex in man are representative examples. Syrian hamsters, as described in this report, do not make alloantibodies to antigens of this type and only very poorly do they carry out in vitro cell-mediated cytotoxicity to target cells putatively bearing these antigens. Since hamsters are quite capable of discriminating analogous antigenic differences in xenogeneic species, and xenogeneic sources cannot distinguish immunologically between the antigens encoded by the two hamster major histocompatibility alleles. Hm-1a and Hm-1b, we conclude that the hamster strains we work with are serologically indistinguishable by the methods used here. However, they obviously differ for determinants which elicit T cell-mediated responses, as evidenced by their ability to express acute skin graft rejection, mixed lymphocyte reactivity, graft-vs-host reactions, and cell-mediated cytotoxicity reactions. Such alloreactivity may reflect a mutation at an SD locus, affecting antigenic sites recognized only by T cells, or that the available hamster strains are SD identical, but differ at loci similar to the I region loci in mice. Alternatively, we cannot exclude the possibility that Syrian hamsters somehow fail to express properly the genes coding for SD determinants.     

Contact reactions between young colonies of the coral Pocillopora damicornis

 Newly settled larvae (primary polyps) or young colonies of the coral Pocillopora damicornis were brought into contact at various periods after planulation to examine isogeneic and allogeneic responses. While young colonies derived from the same colony always fused, those derived from different colonies showed either fusion, nonfusion, or incompatible fusion. Tissues were continuous in incompatibly fused pairs, but a white zone, without zooxanthellae, was observed at the interface. The skeleton was also continuous but a groove with skeletal spines on both sides was observed under the white zone. Polyps originating near the white zone later disappeared or were partially resorbed. After 2–8 months, several incompatibly fused pairs became separated by a skeletal ridge, or by a narrow zone of skeleton without living tissue. Incompatible fusion appears to be a distinct histoincompatible response which later transforms into nonfusion. The period between planulation and initial contact of colonies did not affect the outcomes of the contact experiments. Accepted: 31 January 1996