John P. Cannon

The African coelacanth genome provides insights into tetrapod evolution.

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

Reconstructing immune phylogeny: new perspectives.

Numerous studies of the mammalian immune system have begun to uncover profound interrelationships, as well as fundamental differences, between the adaptive and innate systems of immune recognition. Coincident with these investigations, the increasing experimental accessibility of non-mammalian jawed vertebrates, jawless vertebrates, protochordates and invertebrates has provided intriguing new information regarding the likely patterns of emergence of immune-related molecules during metazoan phylogeny, as well as the evolution of alternative mechanisms for receptor diversification. Such findings blur traditional distinctions between adaptive and innate immunity and emphasize that, throughout evolution, the immune system has used a remarkably extensive variety of solutions to meet fundamentally similar requirements for host protection.

Identification of diversified genes that contain immunoglobulin-like variable regions in a protochordate

The evolutionary origin of adaptive immune receptors is not understood below the phylogenetic level of the jawed vertebrates. We describe here a strategy for the selective cloning of cDNAs encoding secreted or transmembrane proteins that uses a bacterial plasmid (Amptrap) with a defective β-lactamase gene. This method requires knowledge of only a single target motif that corresponds to as few as three amino acids; it was validated with major histocompatibility complex genes from a cartilaginous fish. Using this approach, we identified families of genes encoding secreted proteins with two diversified immunoglobulin-like variable (V) domains and a chitin-binding domain in amphioxus, a protochordate. Thus, multigenic families encoding diversified V regions exist in a species lacking an adaptive immune response.

The phylogenetic origins of the antigen-binding receptors and somatic diversification mechanisms

Summary:  The adaptive immune system arose in ancestors of the jawed vertebrates approximately 500 million years ago. Homologs of immunoglobulins (Igs), T‐cell antigen receptors (TCRs), major histocompatibility complex I (MHC I) and MHC II, and the recombination‐activating genes (RAGs) have been identified in all extant classes of jawed vertebrates; however, no definitive homolog of any of these genes has been identified in jawless vertebrates or invertebrates. RAG‐mediated recombination and associated junctional diversification of both Ig and TCR genes occurs in all jawed vertebrates. In the case of Igs, somatic variation is expanded further through class switching, gene conversion, and somatic hypermutation. Although the identity of the ‘primordial’ receptor that was interrupted by the recombination mechanism in jawed vertebrates may never be established, many different families of genes that exhibit predicted characteristics of such a receptor have been described both within and outside the jawed vertebrates. Recent data from various model systems point toward a continuum of immune receptor diversity, encompassing many different families of recognition molecules whose functions are integrated in an organisms response to pathogenic invasion. Various approaches, including both genomic and protein‐functional analyses, currently are being applied in jawless vertebrates, protochordates, and other invertebrate deuterostome systems and may yield definitive evidence regarding the presence or absence of adaptive immune homologs in species lacking adaptive immune systems. Such studies have the potential for uncovering previously unknown mechanisms of generating receptor diversity.

Induction of myelodysplasia by myeloid-derived suppressor cells

Myelodysplastic syndromes (MDS) are age-dependent stem cell malignancies that share biological features of activated adaptive immune response and ineffective hematopoiesis. Here we report that myeloid-derived suppressor cells (MDSC), which are classically linked to immunosuppression, inflammation, and cancer, were markedly expanded in the bone marrow of MDS patients and played a pathogenetic role in the development of ineffective hematopoiesis. These clonally distinct MDSC overproduce hematopoietic suppressive cytokines and function as potent apoptotic effectors targeting autologous hematopoietic progenitors. Using multiple transfected cell models, we found that MDSC expansion is driven by the interaction of the proinflammatory molecule S100A9 with CD33. These 2 proteins formed a functional ligand/receptor pair that recruited components to CD33’s immunoreceptor tyrosine-based inhibition motif (ITIM), inducing secretion of the suppressive cytokines IL-10 and TGF-β by immature myeloid cells. S100A9 transgenic mice displayed bone marrow accumulation of MDSC accompanied by development of progressive multilineage cytopenias and cytological dysplasia. Importantly, early forced maturation of MDSC by either all-trans-retinoic acid treatment or active immunoreceptor tyrosine-based activation motif–bearing (ITAM-bearing) adapter protein (DAP12) interruption of CD33 signaling rescued the hematologic phenotype. These findings indicate that primary bone marrow expansion of MDSC driven by the S100A9/CD33 pathway perturbs hematopoiesis and contributes to the development of MDS.

Specific lipid recognition is a general feature of CD300 and TREM molecules

CD300, triggering receptor expressed on myeloid cells (TREM), and TREM-like (TREML) receptors are important regulators of the mammalian immune response. Homologs of these receptors, which occur in activating and inhibitory transmembrane forms as well as soluble variants, are found throughout the jawed vertebrates. Specific ligands for most members of these receptor families remain elusive. We report here that at least 11 separate receptors from the CD300, TREM, and TREML families engage in robust and specific interactions with major polar lipids found in prokaryotic and eukaryotic cell membranes. Both soluble and membrane-bound receptor forms exhibit lipid interactions in the solid phase as well as in a physiological signaling context. Overlapping but distinctive patterns of receptor specificity suggest that the CD300/TREM system as a whole may discriminate immunological stimuli based on lipid signatures, thereby influencing downstream responses.

Individual protochordates have unique immune-type receptor repertoires

Innate immunity is mediated by a variety of different, non-rearranging receptors and soluble molecules that recognize and facilitate the elimination of a wide range of pathogens [1]. Immunoglobulin (Ig)-type variable (V) region-containing chitin-binding proteins (VCBPs) found in the protochordate amphioxus, which diverged from the vertebrate lineage before the emergence of adaptive immunity, show structural characteristics of innate immune receptors [2]. Here we describe a very high degree of regionalized hypervariability in one family of VCBPs at the level of the individual germline, producing a unique repertoire of proteins in every animal so far analyzed. In species with large populations, such as amphioxus, extensive polymorphisms may compensate for the absence of somatic modification in the maintenance of immune receptor diversity. The diversified VCBPs in the amphioxus Branchiostoma floridae, a cephalochordate, are soluble proteins consisting of two Ig-type V regions joined to a carboxy-terminal chitin-binding domain [2]. Although their ligands are unknown, VCBPs are likely candidates for innate immune receptors and represent the only example of an innate receptor in which the functional unit is a hyperdiversified Ig-type V region. VCBPs are distributed in at least five families and are expressed specifically and abundantly in the gut. Regionalized peptide sequence hypervariability was noted in the V regions of pooled amphioxus VCBP2 cDNAs [2]. The hypervariable region is centered ~18 residues amino-terminal to the first intradomain cysteine and does not correspond to any of the known complementarity-determining regions of the rearranging antigen-binding receptors found in jawed vertebrates. However, the basis for the hypervariation is not clear. The hypervariable region of the amino-terminal V region of VCBP2 has been characterized in genomic DNA from individual animals collected in the same local geographical area. The germline of every animal encodes a unique VCBP2 receptor repertoire. In a parallel investigation, a 17-fold representative bacterial artificial chromosome library (CHORI-302) constructed from a single reference animal was screened and six VCBP2 family genes were identified, consistent with previous Southern blot analyses [2]. In sum, a total of 43 different peptides are encoded across the amplified region of VCBP2 by only 13 different animals. The most pronounced differences occur across a segment of ~12–15 residues within a 23–30 residue-encoding amplicon (Figure 1). In the majority of cases, any two animals share no more than two specific VCBP2 hypervariable sequences; however, four pairs of animals share three specific hypervariable sequences. Amplicons encoding more conserved regions of the amino-terminal V region …

Ancient evolutionary origin of diversified variable regions demonstrated by crystal structures of an immune-type receptor in amphioxus

Although the origins of genes encoding the rearranging binding receptors remain obscure, it is predicted that their ancestral forms were nonrearranging immunoglobulin-type domains. Variable region–containing chitin-binding proteins (VCBPs) are diversified immune-type molecules found in amphioxus (Branchiostoma floridae), an invertebrate that diverged early in deuterostome phylogeny. To study the potential evolutionary relationships between VCBPs and vertebrate adaptive immune receptors, we solved the structures of both a single V-type domain (to 1.15 Å) and a pair of V-type domains (to 1.85 Å) from VCBP3. The deduced structures show integral features of the ancestral variable-region fold as well as unique features of variable-region pairing in molecules that may reflect characteristics of ancestral forms of diversified immune receptors found in modern-day vertebrates.

New insights into alternative mechanisms of immune receptor diversification.

The clonal commitment, selection, and expansion of B and T lymphocytes expressing diversified receptors provide the underlying basis for the jawed vertebrates adaptive immune response. At the core of this process is the rearrangement and somatic modification of segmental genetic elements that encode the constituent components of immunoglobulins and T-cell antigen receptors. No evidence has been found for a similar mechanism outside of jawed vertebrates; however, invertebrates and jawless vertebrates are subjected to continuous exposure to pathogenic bacteria, viruses, and parasites. The invertebrates and jawless vertebrates as well as jawed vertebrates all encode a variety of mediators of innate immunity. Several reports of extensive germline diversification of conventional innate receptors, as well as molecules that resemble innate receptors but undergo germline and somatic modification, have been made recently. The range of such molecules, which include the fibrinogen-related proteins (FREPs) in a mollusc, variable region-containing chitin-binding proteins (VCBPs) in a cephalochordate, variable lymphocyte receptors (VLRs) in jawless vertebrates, and novel immune-type receptors (NITRs) in bony fish, encompasses both the immunoglobulin gene superfamily (IgSF) and leucine-rich repeat (LRR) proteins. Although these molecules vary markedly in form and likely in function, growing evidence suggests that they participate in various types of host defense and thereby represent significant alternatives to current paradigms of innate and adaptive immune receptors. Unusual genetic mechanisms for diversifying recognition proteins may be a widespread characteristic of animal immunity.

A bony fish immunological receptor of the NITR multigene family mediates allogeneic recognition.

Novel immune-type receptors (NITRs) comprise an exceptionally large, diversified family of activating and inhibitory receptors that has been identified in bony fish. Here, we characterized the structure of an activating NITR that is expressed by a cytotoxic natural killer (NK)-like cell line and that specifically binds an allogeneic B cell target. A single amino acid residue within the NITR immunoglobulin variable (V)-type domain accounts for specificity of the interaction. Structures solved by X-ray crystallography revealed that the V-type domains of NITRs form homodimers resembling rearranging antigen-binding receptor heterodimers. CDR1 elements of both subunits of NITR dimers form ligand-binding surfaces that determine specificity for the nonself target. In the evolution of immune function, it appears that a specific NK type of innate recognition may be mediated by a complex germline multigene family of V structures resembling those that are somatically diversified in adaptive immunological responses.