Michèle Courtet

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.

CTX, a Xenopus thymocyte receptor, defines a molecular family conserved throughout vertebrates

CTX, a cortical thymocyte marker in Xenopus, is an immunoglobulin superfamily (Igsf) member comprising one variable and one constant C2‐type Igsf domain, a transmembrane segment and a cytoplasmic tail. Although resembling that of the TCR and immunoglobulins, the variable domain is not encoded by somatic rearrangement of the gene but by splicing of two half‐domain exons. The C2 domain, also encoded by two exons, has an extra pair of cysteines. The transmembrane segment is free of charged residues, and the cytoplasmic tail (70 amino acids) contains one tyrosine and many glutamic acid residues. ChT1, a chicken homologue of CTX, has the same structural and genetic features, and both molecules are expressed on the thymocyte surface. We cloned new mouse (CTM) and human (CTH) cDNA and genes which are highly homologous to CTX/ChT1 but not lymphocyte specific. Similarity with recently described human cell surface molecules, A33 antigen and CAR (coxsackie and adenovirus 5 receptor), and a number of expressed sequence tags leads us to propose that CTX defines a novel subset of the Igsf, conserved throughout vertebrates and extending beyond the immune system. Strong homologies within vertebrate sequences suggest that the V and C2 CTX domains are scions of a very ancient lineage.

B‐cell development in the amphibian Xenopus

Summary: The amphibian Xenopus and mammals have similar organization and usage of their immunoglobulin gene loci with combinatorial joining of V, D and J elements. The differences in B‐cell development between mammals and this amphibian are due to major differences in developmental kinetics, cell number and lymphoid organ architecture. Unlike mammals, the immune system of Xenopus develops early under pressure to develop quickly and to produce a heterogeneous repertoire before lymphocyte numbers reach 5,000, thereby imposing a limitation on clonal amplification. In addition, it is submitted to metamorphosis. Thus, during the early antigen‐independent period, several features of B‐cell development related to immune diversification are under strict genetically preprogramed control: 1) D reading frames contribute complementary determining region 3 with features that occur in mammals by somatic selection, 2) the temporal stepwise utilization of VH genes in Xenopus occur in families probably because of structural DNA features rather than their position in the locus. Larval and adult immune responses differ in heterogeneity. Larval rearrangements lack N diversity. During the course of immune responses, somatic mutants are generated at the same rate as in other vertebrates but are not optimally selected, probably due to the simpler organization of the lymphoid organs, with neither lymph nodes nor germinal centers resulting in poor affinity maturation. Switch from IgM to other isotypes is mediated by loop‐excision deletion of the IgM constant region gene via switch regions which, unlike their mammalian counterpart, are A‐T rich and reveal conserved microsites for the breakpoints.

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.

Major histocompatibility complex and immunoglobulin loci visualized by in situ hybridization on Xenopus chromosomes.

A technique for fluorescent in situ hybridization (FISH) on chromosomes of the amphibian Xenopus laevis is described. Positive results were obtained with cDNA probes of about 1kb when at least three adjacent copies of the gene are present. The immunoglobulin heavy chain locus is in the centre of the long arm of chromosome 1. Previously, family studies showed that bona fide MHC class Ib genes segregated independently. Now we show that MHC class II alpha and beta genes and class Ib genes are on the same acrocentric chromosome, with MHC in the middle of the long arm, the class Ib complex (XNC) at the tip or the same arm. Each locus or complex is found on only one pair of chromosomes confirming the diploidization of these genes in the pseudotetraploid X. laevis.

Genetic basis of the antibody repertoire in Xenopus: analysis of the Vh diversity.

The Xenopus IgH locus includes various variable (VH) families, several putative diversity (DH) and at least seven joining (JH) elements, but‐‐although structurally very similar to the mammalian locus‐‐it contributes to a restricted antibody repertoire. The largest three VH families contain 15‐30 VH elements which are interspersed at the VHI‐VHII and VHII‐VHIII boundaries. Twenty‐nine genomic and eight expressed VH regions have been sequenced. Each VH family has distinct promoter elements with up to three octamers (ATGCCTAAAT) in either orientation. The incidence of pseudogenes ranges from less than 15% in VHI and VHII to approximately 50% in VHIII, consistent with their relative expression. CDR1 and CDR2 show low overall diversity with nucleotide divergence limited to parts of the CDRs. Randomly selectedly VH elements share CDR1 and CDR2, in some cases also with expressed VH regions. Thus, the complexity of VH elements is not maximal. Patterns of sequence similarities or identities indicate recombination or gene conversion events; sets of direct and inverted repeats flank the sites of, or lie within FR or CDR sequences where these genetic events may occur. Restricted antibody diversity in Xenopus seems therefore to be at least partially related to low complexity of VH elements, frequence of pseudogenes and expression regulated by specific promoter elements; diversity may potentially be increased by (non)homologous recombination events.

Development of the early B cell population in Xenopus

Like mammals, the amphibian Xenopus uses combinatorial joining of the immunoglobulin V, D and J elements and multiple rearrangements to generate its B cell repertoire. Xenopus larvae hatch 2 days after fertilization and individuals are under pressure to develop an immune repertoire when the number of available cells is small (approximately 5 and 200 IgM‐positive cells on days 5 and 11 after fertilization, respectively). In the liver, in a first phase of differentiation spanning days 5 – 12 after fertilization before immunological competence, the heavy (H) chain locus starts rearranging followed by the light (L) chain locus 3 days later. By immunohistology the first B cells expressing H and L chain are detectable on day 10. Despite the small number of cells available and the lack of external antigen selection at these early stages, the repertoire is heterogeneous. The VH families are used stepwise, although their genes are interspersed in the genome. The earliest family used (VH1) is homologous to the VH3 family of human and to the VH7183 of the mouse which are also overrepresented in early mammalian development. In the second phase, from day 12 – 13 onwards, the spleen differ entiates and the animal becomes immunologically competent. The V, D and J usage is similar to that of adults although VDJ junctions lack N nucleotides until metamorphosis. A preferential reading frame for D and one specific DJ junction are overrepresented during this second phase. The visible bias toward homology‐based junction results in fact from selection after rearrangement.

DUPLICATION AND MHC LINKAGE OF THE CTX FAMILY OF GENES IN XENOPUS AND IN MAMMALS

The effects of whole genome duplications that characterize the evolution of vertebrates have been studied on the gene of the Xenopus thymocyte molecule CTX and its mammalian relatives. CTX, with an extracellular part consisting of one V and one C2 external domain, defines a new subset of the immunoglobulin superfamily and is conserved from amphibians to mammals. The number of CTX loci, their polymorphism, and their genetic linkages have been studied in several Xenopus species and in humans. In the genetically simplest species, X. tropicalis (2n = 20), the unique CTX locus is linked to the MHC. In the polyploid species, all CTX genes, unlike many other immune system genes, have remained in the genome; i.  e. there are two CTX loci in the tetraploid species X. laevis (2n = 6) and six CTX loci in the dodecaploid species X. ruwenzoriensis (2n = 108). In X. laevis, one CTX gene is linked to the MHC and the other not, presumably because one set of MHC class I and II has been deleted from the corresponding linkage group. The various mammalian homologues are less related to each other than are the Xenopus CTX genes among each other, and they do not cross‐hybridize with each other because they stem from the ancient polyploidization. Some human CTX homologies are on chromosomes 11 and 21, but others are on chromosomes 1, 6 and 19, which contain MHC paralogous regions; this suggests that a very ancient linkage group has been preserved.

A Xenopus lymphoid tumor cell line with complete Ig genes rearrangements and T-cell characteristics

The first lymphoid cell line derived from an amphibian (Xenopus) thymic tumor shows an extreme form of lineage infidelity. Although it has rearranged in-frame the two alleles of the heavy chain, deleted one light chain locus, and rearranged abortively the two alleles of the second light chain locus, the cell line does not produce immunoglobulin molecules or message. It expresses a variety of T-cell characteristic markers such as Xenopus pan T-cell markers, CD8 equivalent and GATA3 transcription factor. It does not express any major histocompatibility complex class I or class II molecules. It resembles some rare types of mammalian leukemias.

Sequences of Cμ and the VH1 Family in LG7, a Clonable Strain of Xenopus, Homozygous for the Immunoglobulin Loci

Twenty-eight heavy-chain variable (VH1) region genes and the immunoglobulin (IgM) heavy-chain constant region of an isogenic Xenopus hybrid, X. laevis/X, gilli, LG7, have been sequenced. The LG7 clone represents the first Xenopus hybrid that is homozygous for the IgH locus. The VH1 family was specifically investigated because VH1 genes are used by the antibodies produced during the Xenopus antidinitrophenol (DNP) response, These VH1 germ-line sequences establish a so-called ”dictionary— that is available for studying somatic hypermutational mechanisms in immunized frogs.