Tolerance induced by grafting semi-allogeneic adult skin to larval Xenopus laevis: Possible involvement of specific suppressor cell activity

Major histocompatibility complex (MHC)-homozygous Xenopus laevis were rendered tolerant to semi-allogeneic antigens by grafting skins of adult frogs during larval stages (larvally induced tolerance), and this tolerant state was compared with the tolerance induced in early thymectomized frogs by the grafting of semi-allogeneic nonlymphoid thymuses (thymus-reconstituted tolerance). In contrast to a total inability of thymus-reconstituted frogs both to reject skins and to exhibit a mixed leukocyte reaction (MLR) against the semi-allogeneic donor, larvally induced tolerant frogs showed a strong MLR against leukocytes of the tolerizing skin donor (split tolerance). Breakdown of the tolerant state in thymus-reconstituted frogs were easily accomplished by inoculation with syngeneic splenocytes, but this breakdown was extremely difficult to achieve in frogs with larvally induced tolerance. The injection of splenocytes from larvally induced tolerant frogs into normal frogs significantly suppressed semi-allogeneic graft rejection in the latter group; no suppression was obtained when splenocytes from thymus-reconstituted frogs were used. In addition, in the thymectomized frogs, recovery of allograft rejection capacity against the pertinent semi-allogeneic antigens were suppressed by the injection of splenocytes from larvally induced tolerant frogs, with the degree of suppression depending on the splenocyte dose. These results indicate that the larvally induced tolerant state is maintained by specifically induced suppressor cells affecting the in vivo allograft response but not the MLR.     

Immunogenetic aspects of in vivo allotolerance induction during the ontogeny of Xenopus laevis

This study describes the ontogeny of allograft immunity in a partially inbred strain of frogs (Xenopus laevis). At various times during the frogs' premetamorphic, perimetamorphic, and postmetamorphic life, major histocompatibility complex (MHC) homozygous strain JJ Xenopus (MHC haplotype j) were grafted with skin from adult donors of defined MHC homozygous (j,f) and heterozygous (j/f f/h) haplotypes. This protocol reveals that destructive allograft reactivity to MHC alloantigens in Xenopus matures slowly and that allotolerance can be induced to such MHC-encoded antigens throughout larval life. The ultimate fate of an MHC disparate transplant (survival or rejection) is dependent on several interacting variables, which include antigen dose, haplotype dose, and the developmental stage of the host frog at the time of transplantation. In contrast, minor H-locus disparate (MHC compatible) grafts never appear to be rejected by hosts grafted at any larval stage.     

Genetic aspects of the tolerance to allografts induced at metamorphosis in the toadXenopus laevis

Genetic aspects of tolerance to allografts induced at metamorphosis in the toadXenopus laevis were studied in one sibship expressing four different major histocompatibility complex (MHC) haplotypes. Tolerance by skin grafting was induced during metamorphosis in thiourea-blocked individuals, a technique that allows prolonged observation of graft behavior at this stage. Four classes of mutually tolerant animals could be determined. The use of antisera recognizing red blood cell antigens segregating with mixed lymphocyte reaction (MLR) haplotypes revealed that the four abovementioned classes corresponded to the four MHC genotypes of the family. The tolerance is, therefore, preferentially induced to antigens not dependent on the MHC. Under certain circumstances tolerance can also be induced to MHC antigens, provided that the animals differ at the level of one MHC haplotype only. Study of MLR during ontogeny suggested that, between sibs, only the two MLR haplotype differences were stimulatory at metamorphosis, whereas in larval and adult animals a one-haplotype difference was enough for stimulation.     

Larval antigen molecules recognized by adult immune cells of inbred Xenopus laevis: two pathways for recognition by adult splenic T cells

During anuran metamorphosis, larval cells of the tadpole are completely eliminated and replaced by adult cells in the corresponding tissues of the frog for the adaptation to terrestrial life from an aquatic life. Before the metamorphic climax, most of the cells have already transformed from larval cells into adult-type cells, but the tail cells remain as larval cells even at the climax stages of metamorphosis. In our previous works, we demonstrated that larval skin grafts are rejected by an inbred strain of adult Xenopus and that the larval cells are recognized and made apoptotic by splenocytes obtained from adults and/or metamorphosing tadpoles in vitro (Y. Izutsu and K. Yoshizato, 1993, J. Exp. Zool. 266, 163-167; Y. Izutsu et al., 1996, Differentiation 60, 277-286). In the present study, it was found that there were two types of larval epidermal cells, classified according to the presence of major histocompatibility complex (MHC); one is the apical cell expressing both MHC classes I and II, and the other is the skein cell, which expresses no MHC. By a Percoll gradient, we were able to separate these two types of cells and examined the proliferative response of adult T cells to each of them. It was revealed that the apical cells (MHC-positive) were recognized directly by adult splenic T cells, whereas the skein cells (MHC-negative) were recognized by the T cells via the antigen presentation by adult splenocytes. Both of these proliferative responses were restricted to MHC class II. This is the first report showing how the larval-specific antigens present in different forms in epidermal cells are recognized as immunological targets by syngeneic adult T lymphocytes.     

Larval antigen molecules recognized by adult immune cells of inbred Xenopus laevis: partial characterization and implication in metamorphosis

It has been shown that larval skin (LS) grafts are rejected by an inbred strain of adult Xenopus, which suggests a mechanism of metamorphosis by which larval cells are recognized and attacked by the newly differentiating immune system, including T lymphocytes. In an attempt to define the larval antigenic molecules that are targeted by the adult immune system, anti-LS antibodies (IgY) were produced by immunizing adult frogs with syngeneic LS grafts. The antigen molecules that reacted specifically with this anti-LS antiserum were localized only in the larval epidermal cells. Of 53 and 59-60 kDa acidic proteins that were reactive with anti-LS antibodies, a protein of 59 kDa and with an isoelectric point of 4.5 was selected for determination of a 19 amino acid sequence (larval peptide). The rat antiserum raised against this peptide was specifically reactive with the 59 kDa molecules of LS lysates. Immunofluorescence studies using these antisera revealed that the larval-specific molecules were localized in both the tail and trunk epidermis of premetamorphic larvae, but were reduced in the trunk regions during metamorphosis, and at the climax stage of metamorphosis were detected only in the regressing tail epidermis. Culture of splenocytes from LS-immunized adult frogs in the presence of larval peptide induced augmented proliferative responses. Cultures of larval tail pieces in T cell-enriched splenocytes from normal frogs or in natural killer (NK)-cell-enriched splenocytes from early thymectomized frogs both resulted in significant destruction of tail pieces. Tissue destruction in the latter was enhanced when anti-LS antiserum was added to the culture. These results indicate that degeneration of tail tissues during metamorphosis is induced by a mechanism such that the larval-specific antigen molecules expressed in the tail epidermis are recognized as foreign by the newly developing adult immune system, and destroyed by cytotoxic T lymphocytes and/or NK cells.     

Antigen-induced suppression: The role of Class I major histocompatibility antigens

The role of Class I major histocompatibility (MHC) antigens in the induction of specific suppression of graft rejection has been investigated. Two experimental transplantation models have been used - fully vascularized heterotopic cardiac allografts in the mouse and fully vascularized orthotopic renal allografts in the rat. Preparations of cells expressing Class I MHC antigens, for example highly purified preparations of rat erythrocytes or platelets or mouse L cells (H2k) transfected with the D locus Class I gene of the b haplotype, LDb-1 cells, were used to pretreat recipients prior to transplantation. The function of the allograft was monitored in order to assess any beneficial effects induced by Class I MHC antigens. The results obtained implicate Class I MHC as important in the induction of specific immunosuppression of vascularized allograft rejection.     

Ecological immunogenetics of life-history traits in a model amphibian

Major histocompatibility complex (MHC) genes determine immune repertoires and social preferences of vertebrates. Immunological regulation of microbial assemblages associated with individuals influences their sociality, and should also affect their life-history traits. We exposed Xenopus laevis tadpoles to water conditioned by adult conspecifics. Then, we analysed tadpole growth, development and survivorship as a function of MHC class I and class II peptide-binding region amino acid sequence similarities between tadpoles and frogs that conditioned the water to which they were exposed. Tadpoles approached metamorphosis earlier and suffered greater mortality when exposed to immunogenetically dissimilar frogs. The results suggest that developmental regulatory cues, microbial assemblages or both are specific to MHC genotypes. Tadpoles may associate with conspecifics with which they share microbiota to which their genotypes are well adapted.     

MHC class I antigens as surface markers of adult erythrocytes during the metamorphosis of Xenopus

An alloantiserum produced against Xenopus MHC class I antigens has been used to distinguish different erythrocyte populations at metamorphosis. By analysis using a fluorescence-activated cell sorter (FACS) analyzer, tadpole (stage 55) and adult erythrocytes have distinct volume differences and tadpole cells have no MHC antigens on the cell surface. Both tadpole and adult erythrocytes express a "mature erythrocyte" antigen marker, recognized by its monoclonal antibody (F1F6). During metamorphosis, immature erythrocytes, at various stages of differentiation, which express adult levels of cell-surface MHC antigens by 12 days after tail resorption, are found in the bloodstream. These immature cells are biosynthetically active, produce adult hemoglobin, and mature by 60 days after the completion of metamorphosis. Percoll gradient-density fractionation has shown that all of the cells in the new erythrocyte series express adult levels of MHC antigens but there is only a gradual increase in the amount of "mature erythrocyte" antigen. Tadpole erythrocytes, which are biosynthetically active during larval stages, produce small amounts of surface MHC antigens before the metamorphic climax and then become metabolically inactive. They are completely cleared from the circulation by 60 days after metamorphosis. Erythrocytes from tadpoles arrested in their development for long periods of time express intermediate levels of MHC antigens, suggesting a "leaky" expression of these molecules in the tadpole cells. The most abundant erythrocyte cell-surface proteins from tadpoles and adults, as judged by two-dimensional gel electrophoresis, are very different.     

Active Suppression of the Allogeneic Histocompatibility Reactions During the Metamorphosis of the Clawed Toad Xenopus

Metamorphosis is a privileged period for the induction of tolerance to allografts. Transfers of lymphocytes from metamorphosing Xenopus into isogenic adults prevented the rejection of a skin graft differing from the adult host by minor histocompatibility antigens. This implies that active suppression is involved at one step of the induction of tolerance to the self-antigens that differentiate at the time of metamorphosis. The reciprocal experiments of preventing tolerance induction by transfer of normal adult cells into metamorphosing animals failed. However, passive transfer of anti-graft immunity in tolerant animals was partially observed, provided that a transfer of primed cells was done simultaneously with the challenging graft. Thus, memory cells are not as sensitive to the suppression as are the cells that respond in a first set reaction.     

Tolerance to antigen-presenting cell-depleted islet allografts is CD4 T cell dependent

Pretreatment of pancreatic islets in 95% oxygen culture depletes graft-associated APCs and leads to indefinite allograft acceptance in immunocompetent recipients. As such, the APC-depleted allograft represents a model of peripheral alloantigen presentation in the absence of donor-derived costimulation. Over time, a state of donor-specific tolerance develops in which recipients are resistant to donor APC-induced graft rejection. Thus, persistence of the graft is sufficient to induce tolerance independent of other immune interventions. Donor-specific tolerance could be adoptively transferred to immune-deficient SCID recipient mice transplanted with fresh immunogenic islet allografts, indicating that the original recipient was not simply "ignorant" of donor antigens. Interestingly, despite the fact that the original islet allograft presented only MHC class I alloantigens, CD8+ T cells obtained from tolerant animals readily collaborated with naive CD4+ T cells to reject donor-type islet grafts. Conversely, tolerant CD4+ T cells failed to collaborate effectively with naive CD8+ T cells for the rejection of donor-type grafts. In conclusion, the MHC class I+, II- islet allograft paradoxically leads to a change in the donor-reactive CD4 T cell subset and not in the CD8 subset. We hypothesize that the tolerant state is not due to direct class I alloantigen presentation to CD8 T cells but, rather, occurs via the indirect pathway of donor Ag presentation to CD4 T cells in the context of host MHC class II molecules.     

Evolution of the immunomodulatory role of the heat shock protein gp96.

In mammals, certain heat shock proteins (hsps) participate in specialized responses to stressors associated with pathogens or tumors, and as such, act as agents of immune surveillance interacting with both innate and adaptive immunity. We are investigating the conservation of this role throughout the class of vertebrates. We have shown that in Xenopus as in mammals, gp96, the major resident of the endoplasmic reticulum, generates MHC-restricted thymus-dependent immunity in vivo and CR in vitro against minor histocompatibility (H) antigens. By as yet unclear mechanisms that may involve classical MHC-unrestricted cytotoxic CD8+ T cells, gp96 also elicits peptide-specific responses against MHC-class I-negative tumors in adult frogs that may involve cytotoxic NK, MHC-unrestricted CD8+ T and NK/T cells. In naturally MHC class I-deficient but immunocompetent Xenopus larvae, gp96 also generates an innate type of anti-tumor response that is independent of chaperoned peptides. Finally, in a subset of Xenopus sIgM+ B cells, a substantial fraction of gp96 is directed to the cell surface by an active process that is upregulated by bacterial stimulation. This may allow gp96 to access the extracellular compartment without necrosis. Given the dual abilities of gp96 to chaperone antigenic peptides and to modulate innate immune responses, we propose that stimulated B cells that are up-regulating surface gp96 can directly interact with antigen presenting cells (APC) and/or T helper (Th) cells to trigger or amplify immune responses.     

Split tolerance induced by chick embryo thymic epithelium allografted to embryonic recipients

To test the capacity of the epithelial component of the chick embryo thymus to induce tolerance to major histocompatibility complex (MHC) antigens, pre-colonized thymic rudiments were grafted into chick embryonic recipients. Semi-allogeneic or allogeneic transplantations were done between two lines of chickens histocompatible at the MHC locus. Approximately 10% of these thymic chimeras hatched and were studied 3 mo after hatching. Thymic grafts were not rejected by the allogeneic host. The tolerance of chimeric chickens to thymus donor MHC antigens was tested by using a skin graft rejection test and a graft-vs-host (GvH) assay. Chimeric chickens that received an MHC-incompatible thymic graft during the embryonic life tolerated skin graft with the MHC haplotype of the thymus donor. Nevertheless, the lymphocytes within the thymic graft, the host thymus, and the blood were tolerant to the host MHC antigens but were alloreactive in GvH reaction for the MHC antigens of the thymic graft type. These results suggest that the epithelial component of the thymus when taken before the starting of the colonization by hemopoietic precursors and grafted into an early chick embryonic host can induce a tolerance for the MHC determinants involved in allograft rejection but not in the GvH reaction.     

The major histocompatibility complex of primates

The major histocompatibility complex (MHC) encodes cell surface glycoproteins that function in self-nonself recognition and in allograft rejection. Among primates, the MHC has been well defined only in the human; in the chimpanzee and in two species of macaque monkeys the MHC is less well characterized. Serologic, biochemical and genetic evidence indicates that the basic organization of the MHC linkage group has been phylogenetically conserved. However, the number of genes and their linear relationship on the chromosomes differ between species. Class I MHC loci encode molecules that are the most polymorphic genes known. These molecules are ubiquitous in their tissue distribution and typically are recognized together with nominal antigens by cytotoxic lymphocytes. Class II MHC loci constitute a smaller family of serotypes serving as restricting elements for regulatory T lymphocytes. The distribution of class II antigens is limited mainly to cell types serving immune functions, and their expression is subject to up and down modulation. Class III loci code for components C2, C4 and Factor B (Bf) of the complement system.Interspecies differences in the extent of polymorphism occur, but the significance of this finding in relation to fitness and natural selection is unclear. Detailed information on the structure and regulation of MHC gene expression will be required to understand fully the biologic role of the MHC and the evolutionary relationships between species. Meanwhile, MHC testing has numerous applications to biomedical research, especially in preclinical tissue and organ transplantation studies, the study of disease mechanisms, parentage determination and breeding colony management. In this review, the current status of MHC definition in nonhuman primates will be summarized. Special emphasis is placed on the CyLA system of M. fascicularis which is a major focus in our laboratory. A highly polymorphic cynomolgus MHC has been partially characterized and consists of at least 14 A locus, 11 B locus, 7 C locus class I allelic specificities, 9 Ia-like class II antigens and 6 Bf (class III) variants.     

Minor histocompatibility antigen-specific MHC-restricted CD8 T cell responses elicited by heat shock proteins

In mammals, the heat shock proteins (HSP) gp96 and hsp70 elicit potent specific MHC class I-restricted CD8(+) T cell (CTL) response to exogenous peptides they chaperone. We show in this study that in the adult frog Xenopus, a species whose common ancestors with mammals date back 300 million years, both hsp70 and gp96 generate an adaptive specific cellular immune response against chaperoned minor histocompatibility antigenic peptides that effects an accelerated rejection of minor histocompatibility-locus disparate skin grafts in vivo and an MHC-specific CD8(+) cytotoxic T cell response in vitro. In naturally class I-deficient but immunocompetent Xenopus larvae, gp96 also generates an antitumor immune response that is independent of chaperoned peptides (i.e., gp96 purified from normal tissue also generates a significant antitumor response); this suggests a prominent contribution of an innate type of response in the absence of MHC class I Ags.     

The genetic origin of minor histocompatibility antigens

The purpose of this study was to elucidate the genetic origin of minor histocompatibility (H) antigens. Toward this end common inbred mouse strains, distinct subspecies, and species of the subgenus Mus were examined for expression of various minor H antigens. These antigens were encoded by the classical minor H loci H-3 and H-4 or by newly identified minor H antigens detected as a consequence of mutation. Both minor H antigens that stimulate MHC class I-restricted cytotoxic T cells (Tc) and antigens that stimulate MHC class II-restricted helper T cells (Th) were monitored. The results suggested that strains of distinct ancestry commonly express identical or cross-reactive antigens. Moreover, a correlation between the lack of expression of minor H antigens and ancestral heritage was observed. To address whether the antigens found on unrelated strains were allelic with the sensitizing minor H antigens or a consequence of antigen cross-reactivity, classical genetic segregation analysis was carried out. Even in distinct subspecies and species, the minor H antigens always mapped to the site of the appropriate minor H locus. Together the results suggest: 1 minor H antigen sequences are evolutionarily stable in that their pace of antigenic change is slow enough to predate subspeciation and speciation; 2 the minor H antigens originated in the inbred strains as a consequence of a rare polymorphism or loss mutation carried in a founder mouse stock that caused the mouse to percieve the wild-type protein as foreign; 3 there is a remarkable lack of antigenic cross-reactivity between the defined minor H antigens and other products.     

Transgene number-dependent, gene expression rate-independent rejection of Dd -, Kd-, or Dd Kd -transgened mouse skin or tumor cells from C57BL/6 Db Kb mice

Recipient cells migrating into the transplantation site of an allograft recognize histocompatibility antigens on the grafts and are cytotoxic against the grafts. Although the alloreactive immune response is predominantly directed at the major histocompatibility complex (major histocompatibility complex [MHC]; H-2 in mice) class I molecules, the basic mechanisms of allograft rejection (e.g., ligand-receptor interaction) remain unclear, because of the polymorphism and complexity of the MHC. To examine the role of MHC class I molecules in allograft rejection, D(d) , K(d) or D(d) K(d) -transgenic skin or tumor cells we established on a C57BL/6 (D(b) K(b) ) background and transplanted into C57BL/6 mice. Skin grafts from allogeneic (i.e., BALB/c, B10.D2, and BDF1) strains of mice were rejected from C57BL/6 mice on days 12-14 after grafting, whereas isografts were tolerated by these mice. Unexpectedly, skin grafts from D(d) -, K(d) -, and D(d) K(d) -transgenic C57BL/6 mice were rejected on days 12-14 in a transgene expression rate-independent manner from 9/19 (47%), 20/39 (51%), and 12/17 (71%) of C57BL/6 mice, respectively. Similarly, intradermally transplanted allogeneic (i.e., Meth A), but not syngeneic (i.e., EL-4), tumor cells were rejected from C57BL/6 mice; the growth of D(d) - or K(d) -transfected EL-4 cells was delayed by 10-13 days; and 4/10 (40%) of D(d) K(d) -transfected tumor cells were rejected from C57BL/6 mice. These results indicate that D(d) and K(d) genes are equivalent as allogeneic MHC class I genes and that C57BL/6 (D(b) K(b) ) mice reject D(d) -, K(d) -, or D(d) K(d) -transgened skin or tumor cells in a transgene number-dependent, gene expression rate-independent manner.     

Differential expression of creatine kinase isozymes during development of Xenopus laevis: An unusual heterodimeric isozyme appears at metamorphosis

The creatine kinase (CK) repertoire of Xenopus laevis, which is more complex than that of most other vertebrates, involves at least four genomic loci, all showing developmental and tissue-specific expression. The differential expression of this multilocus CK isozyme system was investigated by immunohistology. Specific monoclonal antibodies (mAb) against the three cytoplasmic CK isozymes of Xenopus laevis were isolated and characterized. Two of these mAbs, anti-CK-IV (DM16) and anti-CK-III (JRM4), were specific for CK-IV and CK-III subunits respectively, as well as for the corresponding homodimeric isozymes, CK-IV/IV and CK-III/III. Anti-CK-II (MRX7) mAb recognizes CK-II subunits and CK-II/III heterodimers; the homodimeric CK-II/III does not occur. Immunohistological localization on larval and adult tissue sections reveals that CK-IV epitopes, beside a generalized tissue distribution, are especially concentrated in the cytoplasm of some particular cells such as the photoreceptors in the outer segment of the retina, certain nerve cells of the spinal cord and spinal ganglia, and in larval hepatocytes. The CK-III III isozyme is specifically expressed in skeletal muscle, its appearance and accumulation occurring in parallel with myoblast differentiation. The CK-II antigen is detected first at the time of metamorphosis is skeletal muscles, as well as in the heart, eyes and brain. In striated musculature the expression of CK-II subunits during metamorphosis results in almost complete replacement of CK-III/III homodimers by CK-II/III heterodimers, as indicated by the progressive masking of CK-III epitope and the corresponding appearance of CK-II antigen. In the adult eyes, CK-II antigens localize at the same particular site of photoreceptors as do CK-IV antigens. Since that antigen represents a heterodimeric CK-II/III isozyme, this implies the activation of both CK-II and CK-III genes, none of which is expressed in larval retina.     

Production of H-Y Antibody by Female Mice that Fail to Reject Male Skin

WHEN inbred mice are grafted with skin from inbred donors that differ from the recipients only by a single minor histocompatibility antigen, it is commonly observed that some recipients will retain their skin grafts while others will reject them. This is true of incompatibility for H-Y antigen, which is responsible for the rejection of male grafts by otherwise histocompatible inbred females of the same inbred strain¹. Thus in the DBA/2 (DBA) strain, male-to-female skin grafts are rejected by only some recipients; in the C57BL (B6) strain, females always reject male skin; and C3H/An (C3H) females usually accept male skin grafts indefinitely.     

Larval habitat duration and size at metamorphosis in frogs

The relationship between size at metamorphosis and adult size was studied in 12 closely-related species of frog from Malawi (Central Africa). These species of frogs breed in water of different durations, and occupy different habitats as adults. We could demonstrate no correlation between size at metamorphosis and size of adults when frogs were divided into groups on the basis of occupying similar habitats as adults, but when frogs were divided into groups on the basis of similar duration of larval habitat we demonstrated a strong correlation between size at metamorphosis and adult size. Thus we suggest that duration of the larval habitat is a major determinant of size at metamorphosis, with species which breed in the more temporary habitats metamorphosing at smaller size than species which breed in more permanent habitats, but which are of similar size as adults. Such manipulation of the life cycle appears to be adaptive since it results in individuals becoming independent of water earlier when the likelyhood of early loss of larval habitat is high.     

Major histocompatibility complex-encoded class I molecules are absent in immunologically competent Xenopus before metamorphosis

The expression of class I and class II major histocompatibility complex (MHC)-encoded antigens has been examined at various stages of the development of the clawed frog, Xenopus. By immunoprecipitation with alloantisera or xenoantisera from radio-labeled spleen and thymus lysates, and by mixed lymphocyte reaction analysis, it was determined that the same class II molecules are expressed throughout ontogeny. In contrast, by fluorescence on frozen sections of tadpoles and by immunoprecipitation, the class I molecule is not detected in tadpoles, but appears on all tissues at the climax of metamorphosis. Animals maintained as tadpoles for long periods of time by chemical treatment do express class I antigens; thus, their expression can be independent of other biochemical and morphological changes that occur at metamorphosis. Immunofluorescence detects an otherwise uncharacterized MHC-linked alloantigen on tadpole thymic epithelium from the earliest stages of thymus differentiation.