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In mammals, the T-cell receptor (TCR) complex expressed on mature T-cells consists of α/ß or γ/δ clonotypic heterodimers non-covalently associated with four invariant chains forming the CD3 complex (CD3γ, CD3δ, CD3ε and CD3ζ). The TCR is the unit implicated in the antigenic peptide recognition whereas the CD3 subunits present as three different dimers (δ-ε, γ-ε and ζ-ζ) in the receptor complex participate to the signal transduction and are indispensable for the expression of the TCR at the cell surface. We report the cloning, characterization and expression analysis of CD3γ/δ and CD3ε genes in an amphibian urodele, the Mexican axolotl. Amino acid comparisons show that important motifs and residues were preserved between the axolotl CD3 chains and various vertebrate CD3ε, CD3γ, CD3δ and CD3γ/δ chains. During ontogeny, CD3ε transcripts are first detected in the dorsal region of tail-bud embryos before thymus organogenesis. CD3γ/δ transcripts are first detected in the head of 4-week-old larvae. A cross-reactive polyclonal anti-CD3ε antibody was used for the co-immunoprecipitation of the two CD3 proteins of 25 and 29 kDa, respectively, associated with the 90-kDa αß TCR heterodimer.

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BACKGROUND: Very little is known about the immunological responses of amphibians to pathogens that are causing global population declines. We used a custom microarray gene chip to characterize gene expression responses of axolotls (Ambystoma mexicanum) to an emerging viral pathogen, Ambystoma tigrinum virus (ATV). RESULT: At 0, 24, 72, and 144 hours post-infection, spleen and lung samples were removed for estimation of host mRNA abundance and viral load. A total of 158 up-regulated and 105 down-regulated genes were identified across all time points using statistical and fold level criteria. The presumptive functions of these genes suggest a robust innate immune and antiviral gene expression response is initiated by A. mexicanum as early as 24 hours after ATV infection. At 24 hours, we observed transcript abundance changes for genes that are associated with phagocytosis and cytokine signaling, complement, and other general immune and defense responses. By 144 hours, we observed gene expression changes indicating host-mediated cell death, inflammation, and cytotoxicity. CONCLUSION: Although A. mexicanum appears to mount a robust innate immune response, we did not observe gene expression changes indicative of lymphocyte proliferation in the spleen, which is associated with clearance of Frog 3 iridovirus in adult Xenopus. We speculate that ATV may be especially lethal to A. mexicanum and related tiger salamanders because they lack proliferative lymphocyte responses that are needed to clear highly virulent iridoviruses. Genes identified from this study provide important new resources to investigate ATV disease pathology and host-pathogen dynamics in natural populations.

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Until recently, it was believed that urodele amphibians are able to synthesize only two immunoglobulin isotypes, IgM and IgY. We reinvestigated this issue in the Iberian ribbed newt Pleurodeles waltl and reported recently that this urodele expresses at least three isotypes: IgM, IgP and IgY. In this study, we demonstrate that another urodele, Ambystoma mexicanum, has also a third isotype whose amino acid sequence presents the highest homology with the amino acid sequence of Xenopus IgX. This isotype has typical Ig H-chain characteristics, could form multimers and is mainly expressed in mucosal tissues thereby indicating that it is likely the physiological counterpart of Xenopus IgX and mammalian IgA. Interestingly, no IgP could be found in A. mexicanum, in contrast to P. waltl, in which IgX was not found in previous investigations. These data indicate, for the first time, that different families of urodeles can express different immunoglobulin isotypes.

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Mammals and birds have two major populations of T cells, based on the molecular composition and biological properties of their antigen receptors (TCR). alpha beta T cells recognize antigenic peptides linked to major histocompatibility complex (MHC) molecules, and gamma delta T cells recognize native peptide or non-peptide antigens independently of MHC. Very little is known about gamma delta T cells in ectothermic vertebrates. We have cloned and characterized the TCRdelta chains of an urodele amphibian, the Mexican axolotl (Ambystoma mexicanum). The Cdelta domain is structurally similar to its mammalian homologues and the transmembrane domain is very well conserved. Four of the six Valpha regions that can associate with Calpha (Valpha2, Valpha3, Valpha5 and Valpha6) can also associate with Cdelta, but no specific Vdelta regions were found. This suggests that the axolotl TRD locus is nested within the TRA locus, as in mammals, and that this organization has been present in all tetrapod vertebrates and in the common ancestor of Lissamphibians and mammals, for over 400 million years. Two Jdelta regions were identified, but no Ddelta segments were clearly recognized at the Vdelta-Jdelta junctions. This results in shorter and less variable CDR3 loops than in other vertebrates and the size range of the Vdelta-Jdelta junctions is similar to that of mammalian immunoglobulin light chains. Equivalent quantities of TRD mRNA were found in the lymphoid organs, and in the skin and the intestines of normal and thymectomized axolotls. The analysis of several Valpha/delta6-Cdelta and Vbeta7-Cbeta junctions showed that both the TCRdelta and the TCRbeta chains were limited in diversity in thymectomized axolotls.

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We report here the structure of cDNA clones encoding axolotl light chains of the lambda type. A single IGLC gene and eight different potential IGLV genes belonging to four different families were detected. The axolotl Cgamma domain has several residues or stretches of residues that are typically conserved in mammalian, avian, and Xenopus Cgamma, but the KATLVCL stretch, which is well conserved in the Cgamma and T-cell receptor Cbeta domains of many vertebrate species, is not well conserved. All axolotl Vgamma sequences closely match several human and Xenopus Vgamma-like sequences and, although the axolotl Cgamma and Vgamma sequences are very like their tetrapod homologues, they are not closely related to nontetrapod L chains. Southern blot experiments suggested the presence of a single IGLC gene and of a limited number of IGLV genes, and analysis of IGLV-J junctions clearly indicated that at least three of the IGLJ segments can associate with IGLV1, IGLV2, or IGLV3 subgroup genes. The overall diversity of the axolotl Vgamma CDR3 junctions seems to be of the same order as that of mammalian Vgamma chains. However, a single IGLV4 segment was found among the 45 cDNAs analyzed. This suggests that the axolotl IGL locus may have a canonical tandem structure, like the mammalian IGK or IGH loci. Immunofluorescence, immunoblotting, and microsequencing experiments strongly suggested that most, if not all L chains are of the gamma type. This may explain in part the poor humoral response of the axolotl.

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The Mexican axolotl V(H) segments associated with the Igh C mu and C nu isotypes were isolated from anchored PCR libraries prepared from spleen cell cDNA. The eight new V(H) segments found bring the number of V(H) families in the axolotl to 11. Each V(H) had the canonical structural features of vertebrate V(H) segments, including residues important for the correct folding of the Ig domain. The distribution of ser AGC/T (AGY) and TCN codons in axolotl V(H) genes was biased toward AGY in complementarity-determining region-1 (CDR1) and TCN in framework region-1 (FR1); there were no ser residues in the FR2 region. Thus, the axolotl CDR1 region is enriched in DNA sequences forming potential hypermutation hot spots and is flanked by DNA sequences more resistant to point mutation. There was no significant bias toward AGY in CDR2. Southern blotting using family-specific V(H) probes showed restriction fragments from 1 (V(H)9) to 11-19 (V(H)2), and the total number of V(H) genes was 44 to 70, depending on the restriction endonuclease used. The V(H) segments were not randomly used by the H mu and H nu chains; V(H)1, V(H)6, and V(H)11 were underutilized; and the majority of the V(H) segments belonged to the V(H)7, V(H)8, and V(H)9 families. Most of the nine J(H) segments seemed to be randomly used, except J(H)6 and J(H)9, which were found only once in 79 clones.

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Despite the fact that the axolotl (Ambystoma spp. a urodele amphibian) displays a large T-cell repertoire and a reasonable B-cell repertoire, its humoral immune response is slow (60 days), non-anamnestic, with a unique IgM class. The cytotoxic immune response is slow as well (21 days) with poor mixed lymphocyte reaction stimulation. Therefore, this amphibian can be considered as immunodeficient. The reason for this subdued immune response could be an altered antigenic presentation by major histocompatibility complex (MHC) molecules. This article summarizes our work on axolotl MHC genes. Class I genes have been characterized and the cDNA sequences show a good conservation of non-polymorphic peptide binding positions of the alpha chain as well as a high diversity of the variable amino acids positions, suggesting that axolotl class I molecules can present numerous antigenic epitopes. Moreover, class I genes are ubiquitously transcribed at the time of hatching. These class I genes also present an important polylocism and belong to the same linkage group as the class II B gene; they can be reasonably considered as classical class Ia genes. However, only one class II B gene has been characterized so far by Southern blot analysis. As in higher vertebrates, this gene is transcribed in lymphoid organs when they start to be functional. The sequence analysis shows that the peptide binding region of this class II beta chain is relatively well conserved, but most of all does not present any variability in the beta 1 domain in inbred as well as in wild axolotls, presuming a limited antigenic presentation of few antigenic epitopes. The immunodeficiency of the axolotl could then be explained by an altered class II presentation of antigenic peptides, putting into question the existence of cellular co-operation in this lower vertebrate. It will be interesting to analyze the situation in other urodele species and to determine whether our observations in axolotl represent a normal feature in urodele amphibians. But already two different models in amphibians, Xenopus and axolotl, must be considered in our search for understanding immune system and MHC evolution.

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Class I major histocompatibility complex (Mhc) cDNA clones were isolated from axolotl mRNA by polymerase chain reaction (PCR) and by screening a cDNA phage library. The nucleotide and predicted amino acid sequences show definite similarities to the Mhc class Ialpha molecules of higher vertebrates. Most of the amino acids in the peptide binding region that dock peptides at their N and C termini in mammals are conserved. Several amino acids considered to be important for the interaction of beta2-microglobulin with the Mhc alpha chain are also conserved in the axolotl sequence. The fact that axolotl class I A cDNAs are ubiquitously expressed and highly polymorphic in the alpha1 and alpha2 domains suggests the classical nature of axolotl class I A genes.

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Metamorphosis was induced in neotenic axolotls by immersion of the animals in a solution of thyroid hormone. Hematology of the axolotls was examined before, during, and after metamorphosis. There was a transient decrease in numbers of certain white blood cells during metamorphic climax and a permanent shift in the pattern of circulating cells. The eosinophilic granulocyte was the dominating leukocyte type in neotenes and in metamorphosing animals up to midclimax. Lymphocytes and neutrophilic granulocytes (polymorphs) significantly decreased during midclimax. In postmetamorphic axolotls, lymphocytes and polymorphs predominated. The observed decrease of some leukocytes in metamorphosing animals accords with a transient immunosuppression at metamorphic climax. Metamorphosed axolotls showed a humoral immune response (increase in circulating plasma cells) after repeated antigen challenge, whereas neotenic axolotls did not. Alterations in both cellular and humoral immunity are suggested to occur in both young and adult axolotls following experimental induction of metamorphosis.

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We have cloned 36 different rearranged variable regions (V beta) genes encoding the beta-chain of the T cell receptor in an amphibian species, Ambystoma mexicanum (the Mexican axolotl). Eleven different V beta segments were identified, which can be classified into 9 families on the basis of a minimum of 75% nucleotide identity. All the cloned V beta segments have the canonical features of known mammalian and avian V beta, including conserved residues Cys23, Trp34, Arg69, Tyr90, and Cys92. There seems to be a greater genetic distance between the axolotl V beta families than between the different V beta families of any mammalian species examined to date: most of the axolotl V beta s have fewer than 35% identical nucleotides and the less related families (V beta 4 and V beta 8) have no more than 23.2% identity (13.5% at the amino acid level). Despite their great mutual divergence, several axolotl V beta are sequence-related to some mammalian V beta genes, like the human V beta 13 and V beta 20 segments and their murine V beta 8 and V beta 14 homologues. However, the axolotl V beta 8 and V beta 9 families are not significantly related to any other V beta sequence at the nucleotide level and show limited amino acid similarity to mammalian V alpha, V kappa III, or VH sequences. The detection of nine V beta families among 35 randomly cloned V beta segments suggests that the V beta gene repertoire in the axolotl is probably larger than presently estimated.

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Culture supernatants (PHA-SNs) from axolotl splenocytes cultured with phytohemagglutinin-P (PHA) in medium supplemented with bovine serum albumin (BSA) were collected after 1, 2, and 3 days, pooled, treated to remove residual PHA, precipitated with saturated ammonium sulfate, dialyzed, aliquoted, and stored at -20 degrees C. PHA-SNs stimulated proliferation of homologous lymphoblasts, but not resting splenocytes. SDS-PAGE of metabolically labeled PHA-SNs revealed a band between 14 and 21 kDa. This corresponds to the M(r) of the gel fractions with biological stimulatory activity eluted from PHA-SNs. Blasts absorbed significant bioactivity of PHA-SNs whereas freshly harvested splenocytes did not. Although splenocytes cultured in medium supplemented with 1% fetal bovine serum (FBS) did not proliferate in response to PHA, they did secrete a cytokine with lymphoblast growth-promoting activity. Furthermore, PHA-induced lymphoblasts, initially cultured in medium supplemented with 0.25% BSA, could proliferate in response to PHA-SNs in 1% FBS-supplemented medium.

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We previously reported that a primitive vertebrate, the Mexican axolotl (Amphibian, Urodela) synthesizes two classes of immunoglobulins. IgM are present in serum early in the development, and represent the bulk of specific antibody synthesis after an antigenic challenge. IgY occur in the serum later during the development, and are relatively insensitive to immunization. We demonstrate in the present work, using immunofluorescence with specific Mabs, that IgY are expressed in the gut epithelium, as secretory molecules. Secretory IgY are well expressed in the stomach and intestinal mucosae of young animals from 1 month after hatching to the seventh month. Thereafter, IgY progressively disappear from the gut and become readily detectable in the serum of 9-month-old preadult immunologically mature animals. Axolotl IgY are closely associated in the gut to secretory component-like (SC) molecules that are well-recognized by antisera to the SC of different mammalian species. This is the first description, in a primitive tetrapode, of an immunoglobulin class that could be the physiological counterpart of mammalian IgA.

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Comparative analysis of SDS-PAGE patterns of axolotl spleen cells membrane detergent lysates showed important discrepancies between control and thymectomized animals. Among these, a 38-kD protein band, which appeared as a major protein in controls, was not or poorly expressed after thymectomy. A rabbit antiserum (L12) raised against the 38-kD eluted band labeled in indirect immunofluorescence 80-86% of thymocytes and 40-46% of mIg- lymphoid cells in the spleen. The anti-38-kD antibodies stained in Western blotting two antigenically related polypeptides of 38- and 36-kD on splenocyte membrane lysates. Two-dimensional NEPHGE-PAGE analysis indicated that the anti-38-kD antibodies reacted in the spleen with several gathered spots in the 7.8-8.2 pI range, corresponding to 38-36-kD microheterogeneous polypeptides. Most of these spots are not further expressed in thymectomized animals. These results support evidence that the 38-kD surface antigens can be considered as specific surface markers of the axolotl thymus-derived lymphocytes.

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The ontogeny of immunoglobulin (Ig) synthesis was followed at both cellular and serological levels in the Mexican axolotl (Ambystoma mexicanum) using polyclonal antibodies recognizing all Ig molecules and a set of monoclonal antibodies (Mabs) specific for the C mu and Cv heavy Ig chain isotypes and for the light chain constituents shared by IgM and IgY molecules. Clusters of IgM- and of IgY-synthesizing lymphocytes, often located in separate sites, are first present in spleen sections of 7-week-old 25 mm larvae, about one month after differentiation of the spleen anlage (stage 39-40). In 12-week-old 30-35 mm larvae, the relative proportion of IgM- and IgY-synthesizing cells in the spleen is the same as that in adult animals. However, a marked enhancement of the spleen B cell compartment occurs from 5 to 9 months when Ig-positive cells represent about 88% of the lymphocytes population compared to 60% in adults. No structures equivalent to B cell germinal centers were observed at any stage of the spleen differentiation and cells, although often clustered in small groups, remain dispersed in the entire organ. The relative proportions of IgM and IgY B cells throughout the spleen remain constant during development (about 1 IgY+ cell for 5-6 IgM+ cells) and IgM molecules are first detected in the serum of 2.5-month-old larvae. The enhancement of the serum IgM level correlates well with the absolute number of IgM+ cells in the growing spleen. IgY molecules cannot be detected in the serum before the 7th month but their level quickly increases to reach about 60% of the adult value at 10 months. Thyroxine-induced metamorphosis or hyperimmunization of 4- to 6-month-old larvae had no effect upon the temporal expression of the Ig classes in serum.

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The general thinking about the phylogenic distribution of vertebrate Ig classes is that fish and urodele amphibians are only able to synthesize polymeric IgM-like molecules and that the emergence of a new class of LMW Ig occurs for the first time in anouran species. Following immunization of the Mexican axolotl (Ambystoma mexicanum, Amphibia, Urodela) with TNP-SRBC, HMW anti-TNP antibody molecules are only detected. We have previously shown that these polymeric Ig are constituted of 76 kDa H-chains associated to 27-30 kDa L-chains, respectively recognized by MAbs 33.45.1 and 33.101.2. However, the euglobulin fraction purified from normal axolotl serum contains, beside HMW Ig, abundant 172 kDa molecules which are recognized by MAb 33.101.2 in Western blotting in non-reducing conditions but are not labelled with MAb 33.45.1. In the present work, we characterize this 172 kDa molecule as a LMW Ig which differs from the HMW Ig both at the level of the physicochemical and antigenic properties of their H-chain components. This new 11.9 S axolotl Ig presents some similarities with anouran IgY. The detection of IgY-like molecules in urodele amphibian extends the occurrence of at least two antigenically different H-chain isotypes to all the representative modern classes of the Tetrapoda superclass.

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Both juvenile (14-16 week) and adult (18 month) Ambystoma mexicanum reject skin allografts from adult Ambystoma more speedily than they reject such grafts from juvenile axolotls. Donor-specific histocompatibility antigen, prepared from splenocytes, is more effective in inhibiting adult host splenocyte migration when the antigen is prepared from spleen cells from adult, rather than from juvenile Ambystoma. The thymus is fully developed in juvenile Ambystoma, suggesting that the delayed kinetics of rejection of juvenile allografts reflects immaturity in the expression of histocompatibility antigens to donor skin cells.

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Adult axolotls (A. mexicanum) were hyperimmunized with the haptenic determinant, azobenzene-arsonate (ARS). Specific antibodies were isolated from their serum by immune-affinity chromatography on immobilized ARS columns. Analysis of the specific ARS-binding molecules by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that these animals were capable of producing both high molecular-weight (presumably IgM) and low molecular-weight antibodies to the ARS hapten. The low molecular-weight anti-ARS antibody produced by one inbred colony of axolotls did not show any restricted heterogeneity, as assessed by isoelectric focusing. Our results suggest that regulatory events, not the absence of genetic information, may be responsible for the apparent lack of Ig isotype diversity in this species.

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The effects of X-irradiation were studied on the Mexican axolotl antibody synthesis. To reduce the anti-horse red blood cell (HRBC) antibody titers, 150 rd and smaller doses are ineffective, 200-450 rd are increasingly effective, and 700 rd are maximally effective (and lethal). A significant enhancement of the anti-HRBC titers was observed in low doses (50-150 rd X-irradiated animals). This enhancement was also observed when a low X-ray dose was applied only on the thymic areas. In whole body, but thymus area-shielded, 100 rd X-irradiated animals, the anti-HRBC titers were similar to those of the nonirradiated, HRBC-immunized control group. To explain these phenomena, it is suggested that a radiosensitive, adult thymectomy-sensitive and hydrocortisone-sensitive suppressor T cell subpopulation regulates the antibody synthesis in the axolotl.

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