Lineage specification and plasticity in CD19- early B cell precursors.

We describe here three CD19- B cell precursor populations in mouse bone marrow identified using 12-color flow cytometry. Cell transfer experiments indicate lineage potentials consistent with multilineage progenitor (MLP), common lymphoid progenitor (CLP), and B lineage-restricted pre-pro-B (Fr. A), respectively. However, single cell in vitro assays reveal lineage plasticity: lymphoid/myeloid lineage potential for CLP and B/T lineage potential for Fr. A. Despite myeloid potential, recombination activating gene 2 reporter activation is first detected at low levels in most MLP cells, with 95% of CLPs showing 10-fold increased levels. Furthermore, single cell analysis shows that half of CLP and 90% of Fr. A cells contain heavy chain DJ rearrangements. These data, together with expression profiles of lineage-specific genes, demonstrate progressive acquisition of B lineage potential and support an asynchronous view of early B cell development, in which B lineage specification initiates in the MLP/CLP stage, whereas myeloid potential is not lost until the pre-pro-B (Fr. A) stage, and B/T lymphoid plasticity persists until the CD19+ pro-B stage. Thus, MLP, CLP, and Fr. A represent progressively B lineage-specified stages in development, before the CD19+ B lineage-committed pro-B stage.

Immunoglobulins in the eggs of the nurse shark, Ginglymostoma cirratum.

Elasmobranchs, which include the sharks, skates, and rays, emerged over 450 million years ago and are the oldest vertebrates to possess an adaptive immune system. They have evolved diverse reproductive modes, with a variety of physiological adaptations that enhance reproductive success. The nurse shark, Ginglymostoma cirratum, is an aplacental, viviparous elasmobranch in which the egg and its associated vitelline vasculature are the primary route for maternal-embryonic interactions. During gestation, nurse shark embryos hatch from their eggcases and develop free in the uterus, which is flushed regularly with seawater. Similar to higher vertebrates, embryonic and neonatal nurse sharks possess an immune system that is not fully competent. In birds and bony fishes, maternal immunoglobulins (Ig) stored in the egg during oogenesis confer protective immunity to embryos during gestation. However, early research suggested that such transfer of passive immunity does not occur in sharks. To better understand how elasmobranch embryos are protected from waterborne pathogens during this potentially vulnerable time, we have re-examined the existence of Igs in elasmobranch eggs. Using monoclonal antibodies, we establish the presence of two classes of Igs in nurse shark eggs: 7S IgM and IgNAR. The potential transfer of immunoglobulins from elasmobranch eggs is discussed.

Diversity and repertoire of IgW and IgM VH families in the newborn nurse shark.

BACKGROUND: Adult cartilaginous fish express three immunoglobulin (Ig) isotypes, IgM, IgNAR and IgW. Newborn nurse sharks, Ginglymostoma cirratum, produce 19S (multimeric) IgM and monomeric/dimeric IgM1gj, a germline-joined, IgM-related VH, and very low amounts of 7S (monomeric) IgM and IgNAR proteins. Newborn IgNAR VH mRNAs are diverse in the complementarity-determining region 3 (CDR3) with non-templated nucleotide (N-region) addition, which suggests that, unlike in many other vertebrates, terminal deoxynucleotidyl transferase (TdT) expressed at birth is functional. IgW is present in the lungfish, a bony fish sharing a common ancestor with sharks 460 million years ago, implying that the IgW VH family is as old as the IgM VH family. This nurse shark study examined the IgM and IgW VH repertoire from birth through adult life, and analyzed the phylogenetic relationships of these gene families. RESULTS: IgM and IgW VH cDNA clones isolated from newborn nurse shark primary and secondary lymphoid tissues had highly diverse and unique CDR3 with N-region addition and VDJ gene rearrangement, implicating functional TdT and RAG gene activity. Despite the clear presence of N-region additions, newborn CDR3 were significantly shorter than those of adults. The IgM clones are all included in a conventional VH family that can be classified into five discrete groups, none of which is orthologous to IgM VH genes in other elasmobranchs. In addition, a novel divergent VH family was orthologous to a published monotypic VH horn shark family. IgW VH genes have diverged sufficiently to form three families. IgM and IgW VH serine codons using the potential somatic hypermutation hotspot sequence occur mainly in VH framework 1 (FR1) and CDR1. Phylogenetic analysis of cartilaginous fish and lungfish IgM and IgW demonstrated they form two major ancient gene groups; furthermore, these VH genes generally diversify (duplicate and diverge) within a species. CONCLUSION: As in ratfish, sandbar and horn sharks, most nurse shark IgM VH genes are from one family with multiple, heterogeneous loci. Their IgW VH genes have diversified, forming at least three families. The neonatal shark Ig VH CDR3 repertoire, diversified via N-region addition, is shorter than the adult VDJ junction, suggesting one means of postnatal repertoire diversification is expression of longer CDR3 junctions.

Xenopus class II A genes: studies of genetics, polymorphism, and expression.

The amphibian Xenopus laevis has been a central model for the study of evolution of the major histocompatibility complex (MHC). Many of the counterparts of mammalian MHC genes have been identified in Xenopus, facilitating the understanding of MHC structure and function. Herein we characterize X. laevis MHC class II-alpha chain genes. There are three related class II A genes/haplotype in the four commonly used partially inbred strains, all of which linked to the functional MHC. At least two of these genes in the f haplotype encode full-length cDNA clones and a genomic fragment encoding the immunoglobulin-like domain of the third gene was also characterized. The protein structure and domain organization deduced from the two f/f cDNA clones are similar to mammalian MHC class II-alpha chains. Expression of class II A genes is highest in the spleen and intestine, similar to the previously examined tissue distribution of class II B genes. The two highly expressed genes display high sequence diversity among alleles, similar to what has been found in most other species. Surprisingly, transcript sizes of class II A alleles/isotypes are diverse, suggesting that Xenopus class II allelic lineages are very old.

The plasticity of immunoglobulin gene systems in evolution.

The mechanism of recombination-activating gene (RAG)-mediated rearrangement exists in all jawed vertebrates, but the organization and structure of immunoglobulin (Ig) genes, as they differ in fish and among fish species, reveal their capability for rapid evolution. In systems where there can exist 100 Ig loci, exon restructuring and sequence changes of the constant regions led to divergence of effector functions. Recombination among these loci created hybrid genes, the strangest of which encode variable (V) regions that function as part of secreted molecules and, as the result of an ancient translocation, are also grafted onto the T-cell receptor. Genomic changes in V-gene structure, created by RAG recombinase acting on germline recombination signal sequences, led variously to the generation of fixed receptor specificities, pseudogene templates for gene conversion, and ultimately to Ig sequences that evolved away from Ig function. The presence of so many Ig loci in fishes raises interesting questions not only as to how their regulation is achieved but also how successive whole-locus duplications are accommodated by a system whose function in other vertebrates is based on clonal antigen receptor expression.

Unprecedented multiplicity of Ig transmembrane and secretory mRNA forms in the cartilaginous fish.

In most jawed vertebrates including cartilaginous fish, membrane-bound IgM is expressed as a five Ig superfamily (Igsf)-domain H chain attached to a transmembrane (Tm) region. Heretofore, bony fish IgM was the one exception with IgM mRNA spliced to produce a four-domain Tm H chain. We now demonstrate that the Tm and secretory (Sec) mRNAs of the novel cartilaginous fish Ig isotypes, IgW and IgNAR, are present in multiple forms, most likely generated by alternative splicing. In the nurse shark, Ginglymostoma cirratum, and horn shark, Heterodontus francisci, alternative splicing of Tm exons to the second or the fourth constant (C(H)) exons produces two distinct IgW Tm cDNAs. Although the seven-domain IgW Sec cDNA form contains a canonical secretory tail shared with IgM, IgNAR, and IgA, we report a three-domain cDNA form of shark IgW (IgW(short)) having an unusual Sec tail, which is orthologous to skate IgX(short) cDNA. The IgW and IgW(short) Sec transcripts are restricted in their tissue distribution and expression levels vary among individual sharks, with all forms expressed early in ontogeny. IgNAR mRNA is alternatively spliced to produce a truncated four-domain Tm cDNA and a second Tm cDNA is expressed identical in Igsf domains as the Sec form. PBL is enriched in the Tm cDNA of these Igs. These molecular data suggest that cartilaginous fish have augmented their humoral immune repertoire by diversifying the sizes of their Ig isotypes. Furthermore, these Tm cDNAs are prototypical and the truncated variants may translate as more stable protein at the cell surface.

Heterogeneity among DN1 prothymocytes reveals multiple progenitors with different capacities to generate T cell and non-T cell lineages.

The nature of early T lineage progenitors in the thymus or bone marrow remains controversial. Here we assess lineage capacity and proliferative potential among five distinct components of the earliest intrathymic stage (DN1, CD25(-)44(+)). All of these express one or more hemato-lymphoid lineage markers. All can produce T lineage cells, but only two of them display kinetics of differentiation, proliferative capacity, and other traits consistent with being canonical T progenitors. The latter also appeared limited to producing cells of the T or NK lineages, while B lineage potential derived mainly from the other, less typical T progenitors. In addition to precisely defining canonical early progenitors in the thymus, this work reconciles conflicting results from numerous groups by showing that multiple progenitors with a DN1 phenotype home to the thymus and make T cells, but possess different proliferative potentials and lineage capacities.

Terminal deoxynucleotidyl transferases from elasmobranchs reveal structural conservation within vertebrates.

The DNA polymerase (pol) X family is an ancient group of enzymes that function in DNA replication and repair (pol beta), translesion synthesis (pol lambda and pol micro) and terminal addition of non-templated nucleotides. This latter terminal deoxynucleotidyl transferase (TdT) activity performs the unique function of providing diversity at coding joins of immunoglobulin and T-cell receptor genes. The first isolated full-length TdT genes from shark and skate are reported here. Comparisons with the three-dimensional structure of mouse TdT indicate structural similarity with elasmobranch orthologues that supports both a template-independent mode of replication and a lack of strong nucleotide bias. The vertebrate TdTs appear more closely related to pol micro and fungal polymerases than to pol lambda and pol beta. Thus, unlike other molecules of adaptive immunity, TdT is a member of an ancient gene family with a clear gene phylogeny and a high degree of similarity, which implies the existence of TdT ancestors in jawless fishes and invertebrates.

J chain in the nurse shark: implications for function in a lower vertebrate.

J chain is a small polypeptide covalently attached to polymeric IgA and IgM. In humans and mice, it plays a role in binding Ig to the polymeric Ig receptor for transport into secretions. The putative orthologue of mammalian J chain has been identified in the nurse shark by sequence analysis of cDNA and the polypeptide isolated from IgM. Conservation with J chains from other species is relatively poor, especially in the carboxyl-terminal portion, and, unlike other J chains, the shark protein is not acidic. The only highly conserved segment in all known J chains is a block of residues surrounding an N-linked glycosylation site. Of the eight half-cystine residues that are conserved in mammalian J chains, three are lacking in the nurse shark, including two in the carboxyl-terminal segment that have been reported to be required for binding of human J chain-containing IgA to secretory component. Taken together with these data, the relative abundance of J chain transcripts in the spleen and their absence in the spiral valve (intestine) suggest that J chain in nurse sharks may not have a role in Ig secretion. Analysis of J chain sequences in diverse species is in agreement with accepted phylogenetic relationships, with the exception of the earthworm, suggesting that the reported presence of J chain in invertebrates should be reassessed.