L. Du Pasquier

What limits affinity maturation of antibodies in Xenopus--the rate of somatic mutation or the ability to select mutants?

Although the Xenopus immunoglobulin heavy chain locus is structurally and functionally similar to mammalian IgH loci, Xenopus antibodies are limited in heterogeneity, and they mature only slightly in affinity during immune responses. During the antibody response of isogenic frogs to DNP‐KLH, mu and upsilon cDNA sequences using elements of the VH1 family were cloned, sequenced and compared with germline counterparts. There were zero to four mutations per sequence, mostly single base substitutions, in the framework and CDRs 1 and 2 of VH. No mutations were found in JH. Since the point mutation rate was only 4‐ to 7‐fold lower than that calculated for mice, affinity maturation does not seem to be limited by mutant availability. Because of a relatively low ratio of replacement to silent mutations in the CDRs and a very high ratio of GC to AT base pairs altered by mutation, it is suggested that the problem results from the absence of an effective mechanism for selecting mutants, which in turn might be related to the absence of germinal centers in Xenopus.

A novel type of class I gene organization in vertebrates: a large family of non-MHC-linked class I genes is expressed at the RNA level in the amphibian Xenopus.

A Xenopus class I cDNA clone, isolated from a cDNA expression library using antisera, is a member of a large family of non‐classical class I genes (class Ib) composed of at least nine subfamilies, all of which are expressed at the RNA level. The subfamilies are well conserved in their immunoglobulin‐like alpha 3 domains, but their peptide‐binding regions (PBRs) and cytoplasmic domains are very divergent. In contrast to the great allelic diversity found in the PBR of classical class I genes, the alleles of one of the Xenopus non‐classical subfamilies are extremely well conserved in all regions. Several of the invariant amino acids essential for the anchoring of peptides in the classical class I groove are not conserved in some subfamilies, but the class Ib genes are nevertheless more closely related in the PBR to classical and non‐classical genes linked to the MHC in mammals and birds than to any other described class I genes like CD1 and the neonatal rat intestinal Fc receptor. Comparison with the Xenopus MHC‐linked class Ia protein indicate that amino acids presumed to interact with beta 2‐microglobulin are identical or conservatively changed in the two major class I families. Genomic analyses of Xenopus species suggest that the classical and non‐classical families diverged from a common ancestor before the emergence of the genus Xenopus over 100 million years ago; all of the non‐classical genes appear to be linked on a chromosome distinct from the one harboring the MHC. We hypothesize that this class Ib gene family is under very different selection pressures from the classical MHC genes, and that each subfamily may have evolved for a particular function.

A major histocompatibility complex in the toadxenopus laevis (Daudin)

The genetic relationship between mixed leukocyte reaction (MLR), skin graft rejection, and some red blood cell antigens has been studied in a sibship of the toadXenopus laevis. MLR typing was achieved using blood lymphocytes. The graft experiments were performed at 17–19°C. Grafts exchanged between MLR identical sibs were rejected in 30.9±5.1 days, grafts exchanged between MLR different sibs were rejected in 20.4±2.4 days when animals differed at one MLR haplotype, and in 18.6±1.9 days when they differed at two MLR haplotypes. Immunizations and absorptions following the MLR typing produced agglutinating antisera that recognize red blood cell antigens segregating with MLR haplotypes. The results parallel those obtained in various mammalian and avian species and suggest that the homology of the major histocompatibility complex (MHC), described in higher vertebrates, can be extended to amphibians.

Cloning of JAM-2 and JAM-3: an Emerging Junctional Adhesion Molecular Family

Throughout embryonic and early postnatal development, endothelial cells proliferate and differentiate to form new blood vessels via vasculogenesis and angiogenesis (1, 2). In adult organisms the endothelium defines the blood-tissue barrier and consists of non-cycling quiescent cells. These polarized cells are linked to each other by tight junctions and adherens junctions to form a continuous layer of cells. JAM (hereafter referred as JAM-1) and VE-cadherin were characterized as endothelial adhesion molecules participating in tight and adherens intercellular junctions respectively (3, 4). These molecules were shown to regulate vascular functions such as monocyte transmigration or paracellular permeability, probably as a consequence of their structural contribution to the vessel wall. It is well established that the regulated and coordinated expression of adhesion molecules is necessary to maintain normal vascular functions such as tissue homeostasis, vascular permeability, leukocyte emigration, fibrinolysis, coagulation and vasotonus. Temporary changes in the adhesion properties of vascular endothelium have been observed during inflammation, tumor growth, wounding or angiogenesis (5-7). The presence of a growing tumor increases the local concentration of angiogenic factors leading to a switch from non-cycling quiescent endothelial cells to proliferating endothelium. As a result, endothelial cells of existing vessels degrade the extracellular matrix (ECM) and invade the surrounding tissue, which leads to vascularization of tumors. During the angiogenic switch, the pattern of endothelial gene expression is modified, and the level of transcripts encoding protein s involved in cell migration or cell division is affected. For example, the balance between proteases/antiproteases such as PA/PAI1 is changed, leading to increased endothelial cell motility (8). Moreover, the treatment of endothelial cells with angiogenic factors results in a fourfold increase in αVβ3 integrin expression, an adhesion molecule implicated in cell migration (9). In addition, angiogenesis was shown to modify the endothelial inflammatory response leading to abnormal expression of inflammatory adhesion molecules (10, 11). These examples do not constitute an exhaustive list, but support the hypothesis that during the angiogenic switch, the global adhesion behavior of endothelial cells is changed.

The Chicken Leukocyte Receptor Complex: A Highly Diverse Multigene Family Encoding at Least Six Structurally Distinct Receptor Types

The chicken Ig-like receptors (CHIR) have been described as two Ig domain molecules with long cytoplasmic tails containing inhibitory motifs. In this study, we demonstrate that CHIR form a large family, with multiple members showing great sequence variability among members as well as a great diversity in domain organization and properties of the transmembrane and cytoplasmic segments. We characterize various novel receptor types with motifs indicative of inhibitory, activating, or both functions. In addition to the inhibitory receptors with two ITIM, receptors with a single immunoreceptor tyrosine-based switch motif or receptors lacking a cytoplasmic domain were isolated. Activating receptors with a short cytoplasmic domain and a transmembrane arginine assembled with the newly identified chicken FcεRIγ chain. Three bifunctional receptor types were characterized composed of one or two C2-type Ig-like domains, a transmembrane region with a positively charged residue and combinations of cytoplasmic motifs such as ITIM, immunoreceptor tyrosine-based switch motif, and YXXM. RT-PCR revealed distinct expression patterns of individual CHIR. All receptor types shared a conserved genomic architecture, and in single Ig domain receptors a pseudoexon replaced the second Ig exon. Southern blot analyses with probes specific for the Ig1 domain were indicative of a large multigene family. Of 103 sequences from the Ig1 domain of a single animal, 41 unique sequences were obtained that displayed extensive variability within restricted Ig regions. Fluorescence in situ hybridization localized the CHIR gene cluster to microchromosome 31 and identified this region as orthologous to the human leukocyte receptor complex.

Organization and rearrangement of immunoglobulin M genes in the amphibian Xenopus.

Sequences of immunoglobulin (Ig) cDNA clones of Xenopus laevis show that at least three different VH families are expressed in association with different JH elements and different isotypes of Ig constant regions. In genomic Southern blot analysis, the VH probes for each family hybridize to a distinct set of multiple DNA fragments. In contrast, the genomic JH elements and the IgM constant region gene are localized in a single DNA fragment of approximately 15 kb. Genomic VH elements contain regulatory sequences similar to those in VH genes of shark, fish and mammals and have a leader peptide sequence that contains an intron; they encode the VH region until residue 95 and have heptamer–23‐bp–nonamer motifs similar to the rearrangement signal sequences (RSS) in all other vertebrate VH elements. The six genomic JH elements so far sequenced have a nonamer–23‐bp–heptamer motif at their 5′ end. These RSS motifs imply the existence of DH elements. The comparison of cDNA clones that contain similar constant regions but different VH regions or JH elements suggest rearrangement events. This is shown by Southern blot analysis of erythrocyte and B cell DNA with a JH probe. Thus, the overall organization of the Xenopus Ig gene locus is similar to that of mammals but strikingly different from shark.

The ontogeny of diversification at the immunoglobulin heavy chain locus in Xenopus.

Since the larval and adult antibody responses are distinct and restricted in the clawed toad Xenopus, it offers a near ideal model for studying the ontogeny of antibody repertoires and the mechanisms involved. Immunoglobulin heavy chain (IgH) cDNA clones and B cell IgH DNA clones from various larval and adult libraries have been analysed in isogenic Xenopus. Some features are similar in adults and tadpoles, while others differ and explain the particularities observed previously at the protein level. Among the similarities we found are: (i) the mode of rearrangements (there are approximately 50% abortive events in B cells from both stages), (ii) VH family usage (10 of 11 known VH families are expressed proportionally to the number of VH elements per family), and (iii) JH usage (of the eight to nine Xenopus JH elements, two are used in approximately 70% of the VH regions in both stages of development). We found that there is relatively higher membrane exon expression in tadpoles compared with adults; and that most of the differences come from the diversification of CDR3 through DH usage and N diversification. Unlike in mammals, Xenopus DH elements are used with a remarkable flexibility with inversion, fusions and usage in different reading frames, but tadpoles show a strong bias for the usage of only a few DH elements and of a preferred reading frame. There is N diversification, which further increases CDR3 heterogeneity, in adult Xenopus but virtually none in tadpoles. These observations can account for the fact that larval antibody responses are less heterogeneous than those of adults.

The effect of thymectomy on the mixed leukocyte reaction and phytohemagglutinin responsiveness in the clawed toadXenopus laevis

In the clawed toadXenopus laevis, thymectomy at 7 days of age abrogates the phytohemagglutinin response of leukocytes and the mixed leukocyte reaction.

Somatic mutation in ectothermic vertebrates : Musings on selection and origins

The antibody responses of ectothermic (cold blooded) vertebrates do not mature in the same fashion as responses analyzed in mice. This has been demonstrated by either a total lack of affinity maturation in some species or a much lower rise in affinity in others (reviewed in Du Pasquier 1993). When it was determined that all ectotherms studied possess large numbers of V(D)J genes, and that rearrangement processes to establish Ig repertoires are essentially the same as those in mouse and human, it was theorized that poor immune responses in ectotherms could be explained by a suboptimal utilization of somatic mutants (Du Pasquier 1982, 1993). Another interpretation was that somatic mutation in the immune system arose late in vertebrate evolution, after emergence of the rearrangement process that generates functional V genes (Matsunaga 1985). Recent studies in cartilaginous fish (horned shark and nurse shark) and an amphibian (Xenopus) have shown conclusively that in addition to all of the molecular building blocks of the adaptive immune system, somatic hypermutation in immune-related genes is present in all jawed vertebrates. In this chapter we review the evidence for mutation in ectotherms, debate its importance to the immune system of these creatures, and speculate on its origins.

Structural and functional analysis of spontaneous anti-nitrophenyl antibodies in three cyprinid fish species: Carp (Cyrinuscarpio), goldfish (Carassiusauratus) and tench (Tincatinca)

High spontaneous anti-trinitrophenyl (TNP) activities were found in three Cyprinid fish species: Carp (Cyprinuscarpio), Goldfish (Carassiusauratus) and Tench (Tincatinca). The molecules involved, isolated by affinity chromatography on dinitrophenyl-lysine Sepharose (DNP-lysine-Sepharose), had the main charasteristics of a high molecular weight immunoglobulin (IgM-like). Affinity measurements were performed on natural anti-DNP/TNP antibodies isolated from nine individual tench sera, using the inhibition of DNP-T4 bacteriophage inactivation technique. The antibodies analysed were more specific for TNP than for DNP. No activity was found against paranitrophenyl hapten. Affinities were all very low, even for TNP. In the three species, natural anti-DNP/TNP antibodies constitute as much as 11 to 16% of the total immunoglobulin concentration. This high level of nitrophenyl-binding serum immunoglobulins either suggests the existence of a particular regulatory mechanism in fish or reflects a generally low antibody diversity in these species.