Jianxu Li

Dual nature of the adaptive immune system in lampreys

Jawless vertebrates use variable lymphocyte receptors (VLR) comprised of leucine-rich-repeat (LRR) segments as counterparts of the immunoglobulin-based receptors that jawed vertebrates use for antigen recognition. Highly diverse VLR genes are somatically assembled by the insertion of variable LRR sequences into incomplete germline VLRA and VLRB genes. Here we show that in sea lampreys (Petromyzon marinus) VLRA and VLRB anticipatory receptors are expressed by separate lymphocyte populations by monoallelic VLRA or VLRB assembly, together with expression of cytosine deaminase 1 (CDA1) or 2 (CDA2), respectively. Distinctive gene expression profiles for VLRA+ and VLRB+ lymphocytes resemble those of mammalian T and B cells. Although both the VLRA and the VLRB cells proliferate in response to antigenic stimulation, only the VLRB lymphocytes bind native antigens and differentiate into VLR antibody-secreting cells. Conversely, VLRA lymphocytes respond preferentially to a classical T-cell mitogen and upregulate the expression of the pro-inflammatory cytokine genes interleukin-17 (IL-17) and macrophage migration inhibitory factor (MIF). The finding of T-like and B-like lymphocytes in lampreys offers new insight into the evolution of adaptive immunity.

Definition of a third VLR gene in hagfish

Significance The jawless vertebrates (hagfish and lampreys) possess an alternative adaptive immune system in which variable lymphocyte receptors (VLRs) constructed of leucine-rich repeats are used to recognize foreign antigens. Three VLR genes have been identified in lampreys (VLRA, VLRB, and VLRC), but only two (VLRA and VLRB) have been found in hagfish. Here, we identified and characterized a third hagfish VLR gene. Our analysis indicates that the third hagfish VLR is the ortholog of lamprey VLRA, while the previously identified hagfish “VLRA” is the counterpart of lamprey VLRC. The demonstration of three orthologous VLR genes in hagfish and lampreys suggests that this anticipatory receptor system evolved in a common ancestor of the two jawless vertebrate lineages ∼480 Mya. Jawless vertebrates (cyclostomes) have an alternative adaptive immune system in which lymphocytes somatically diversify their variable lymphocyte receptors (VLR) through recombinatorial use of leucine-rich repeat cassettes during VLR gene assembly. Three types of these anticipatory receptors in lampreys (VLRA, VLRB, and VLRC) are expressed by separate lymphocyte lineages. However, only two VLR genes (VLRA and VLRB) have been found in hagfish. Here we have identified a third hagfish VLR, which undergoes somatic assembly to generate sufficient diversity to encode a large repertoire of anticipatory receptors. Sequence analysis, structural comparison, and phylogenetic analysis indicate that the unique hagfish VLR is the counterpart of lamprey VLRA and the previously identified hagfish “VLRA” is the lamprey VLRC counterpart. The demonstration of three orthologous VLR genes in both lampreys and hagfish suggests that this anticipatory receptor system evolved in a common ancestor of the two cyclostome lineages around 480 Mya.

Comparative genomics and evolution of the alpha-defensin multigene family in primates.

Defensin genes encode small cationic antimicrobial peptides that form an important part of the innate immune system. They are divided into three families, alpha (α), beta (β), and theta (), according to arrangement of the disulfide bonding pattern between cysteine residues. Considering the functional importance of defensins, investigators have studied the evolution and the genomic organization of defensin genes. However, these studies have been restricted mainly to β-defensins. To understand the evolutionary dynamics of α-defensin genes among primates, we identified the α-defensin repertoires in human, chimpanzee, orangutan, macaque, and marmoset. The α-defensin genes in primates can be classified into three phylogenetic classes (class I, II, and III). The presence of all three classes in the marmoset indicates that their divergence occurred before the separation of New World and Old World monkeys. Comparative analysis of the α-defensin genomic clusters suggests that the makeup of the α-defensin gene repertoires between primates is quite different, as their genes have undergone dramatic birth-and-death evolution. Analysis of the encoded peptides of the α-defensin genes indicates that despite the overall high level of sequence divergence, certain amino acid residues or motifs are conserved within and between the three phylogenetic classes. The evolution of α-defensins in primates, therefore, appears to be governed by two opposing evolutionary forces. One force stabilizes specific amino acid residues and motifs to preserve the functional and structural integrity of the molecules and the other diversifies the sequences generating molecules with a wide range of activities against a large number of pathogens.

Characterization of Lamprey BAFF-like Gene: Evolutionary Implications

BAFF (TNF superfamily [TNFSF] 13B/Blys) and APRIL (TNFSF13) are important regulatory factors for lymphocyte activation and survival in mammals. A BAFF/APRIL-like relative called BAFF- and APRIL-like molecule (BALM) has also been identified in cartilaginous and bony fishes, and we report in this study a BAFF-like gene in lampreys. Our phylogenetic analysis of these genes and a related TNFSF12 gene called TNF-like weak inducer of apoptosis (TWEAK) suggest that, whereas an ancestral homolog of BAFF and APRIL was already present in a common ancestor of jawed and jawless vertebrates, TWEAK evolved early on in the jawed vertebrate lineage. Like mammalian BAFF and APRIL, the lamprey BAFF-like gene is expressed in T-like, B-like, and innate immune cells. The predicted protein encoded by this BAFF-like gene in lampreys exhibits higher sequence similarity with mammalian BAFF than APRIL. Correspondingly, we find BAFF orthologs in all of the jawed vertebrate representatives that we examined, although APRIL and/or BALM orthologs are not identifiable in certain jawed vertebrates. For example, BALM is not identifiable in tetrapods, and APRIL is not identifiable in several bony fishes or in birds, the latter of which also lack a TWEAK-like gene. Our analysis further suggests that a hybrid molecule called TWE-PRIL, which is a product of an in-genomic fusion between APRIL and TWEAK genes evolved early in mammalian evolution.

Evolution of two prototypic T cell lineages

Jawless vertebrates, which occupy a unique position in chordate phylogeny, employ leucine-rich repeat (LRR)-based variable lymphocyte receptors (VLR) for antigen recognition. During the assembly of the VLR genes (VLRA, VLRB and VLRC), donor LRR-encoding sequences are copied in a step-wise manner into the incomplete germ-line genes. The assembled VLR genes are differentially expressed by discrete lymphocyte lineages: VLRA- and VLRC-producing cells are T-cell like, whereas VLRB-producing cells are B-cell like. VLRA(+) and VLRC(+) lymphocytes resemble the two principal T-cell lineages of jawed vertebrates that express the αβ or γδ T-cell receptors (TCR). Reminiscent of the interspersed nature of the TCRα/TCRδ locus in jawed vertebrates, the close proximity of the VLRA and VLRC loci facilitates sharing of donor LRR sequences during VLRA and VLRC assembly. Here we discuss the insight these findings provide into vertebrate T- and B-cell evolution, and the alternative types of anticipatory receptors they use for adaptive immunity.

Genomic donor cassette sharing during VLRA and VLRC assembly in jawless vertebrates.

Significance Lampreys possess two T-like lymphocyte lineages that express either variable lymphocyte receptor (VLR) A or VLRC antigen receptors. Despite the mutually exclusive expression pattern of VLRA and VLRC, in some cases the sequences of the two receptors are partially identical. This is the result of the shared use of genomic donor cassettes that are required to convert the incomplete VLRA and VLRC genes into functional assemblies. This feature is reminiscent of T-cell receptors of jawed vertebrates that, despite being composed of different molecular structures, also share some variable parts. The shared use of variable segments in the different antigen receptor types for T cells of all vertebrates implies a conserved functional relationship between the two prototypic T-cell lineages. Lampreys possess two T-like lymphocyte lineages that express either variable lymphocyte receptor (VLR) A or VLRC antigen receptors. VLRA+ and VLRC+ lymphocytes share many similarities with the two principal T-cell lineages of jawed vertebrates expressing the αβ and γδ T-cell receptors (TCRs). During the assembly of VLR genes, several types of genomic cassettes are inserted, in step-wise fashion, into incomplete germ-line genes to generate the mature forms of antigen receptor genes. Unexpectedly, the structurally variable components of VLRA and VLRC receptors often possess partially identical sequences; this phenomenon of module sharing between these two VLR isotypes occurs in both lampreys and hagfishes. By contrast, VLRA and VLRC molecules typically do not share their building blocks with the structurally analogous VLRB receptors that are expressed by B-like lymphocytes. Our studies reveal that VLRA and VLRC germ-line genes are situated in close proximity to each other in the lamprey genome and indicate the interspersed arrangement of isotype-specific and shared genomic donor cassettes; these features may facilitate the shared cassette use. The genomic structure of the VLRA/VLRC locus in lampreys is reminiscent of the interspersed nature of the TCRA/TCRD locus in jawed vertebrates that also allows the sharing of some variable gene segments during the recombinatorial assembly of TCR genes.

B Cells and Antibodies in Jawless Vertebrates

Abstract An adaptive immune system (AIS) based on clonally diverse lymphocytes that can recognize specific pathogens and provide protective memory against a second encounter is found only in vertebrate species. Alternative AISs have now been defined in jawed and jawless vertebrates. Both of these systems use lymphocytes with a wide variety of anticipatory receptors, but they differ in that the lymphocytes in jawless vertebrates (lampreys and hagfish) use leucine-rich repeat (LRR)-based variable lymphocyte receptors (VLRs) for antigen recognition, whereas lymphocytes in vertebrates with jaws use immunoglobulin (Ig)-based receptors for the same purpose. There are three types of lamprey VLR genes, VLRA, VLRB, and VLRC, that are assembled and expressed in a clonally diverse manner by separate populations of lymphocytes that resemble our thymus-derived γδ and αβ T lymphocytes and bone marrow-derived B lymphocytes. The parallels between these lymphocyte lineages in jawless and jawed vertebrates indicate that the genetic programs for the major lymphocyte differentiation pathways evolved before the convergent evolution of the VLR and B cell receptor/T cell receptor types of antigen receptors. This chapter focuses on the current understanding of the lamprey B cells and their LRR-based antibody products.