Variable Lymphocyte Receptors Research Articles

Investigation of variable lymphocyte receptors in the alternative adaptive immune response of hagfish

Jawless vertebrates have an alternative adaptive immune system mediated by variable lymphocyte receptors (VLRs), VLRA, VLRC and VLRB. In investigation on the adaptive immunity of hagfish, avian influenza virus hemagglutinin (H9N2-HA1) was used as a model antigen, with mRNA expression levels of VLRA, VLRC and Ikaros were up-regulated in the first week post-immunization. CD45 was up-regulated after the first week; and expression of VLRB progressively increased over the course of the trial. The transcriptional/translational activation of VLRB in blood was verified. The VLRBs cloned from these transcripts showed diversity in their leucine-rich repeats (LRRs). The production of specific VLRB increased in a time- and dose-dependent manner, detected by an anti-VLRB antibody (11G5). The plasma VLRB could distinguish H9N2-HA1 from unrelated proteins, but not from other HA1 subtypes. Together, our findings show that VLRs play a major role in the alternative adaptive immune system of hagfish by responding to specific foreign substances, such as H9N2-HA1.

Selection of specific interactors from phage display library based on sea lamprey variable lymphocyte receptor sequences

Variable lymphocyte receptors (VLRs) are non-immunoglobulin components of adaptive immunity in jawless vertebrates. These proteins composed of leucine-rich repeat modules offer some advantages over antibodies in target binding and therefore are attractive candidates for biotechnological applications. In this paper we report the design and characterization of a phage display library based on a previously proposed dVLR scaffold containing six LRR modules [Wezner-Ptasinska et al., 2011]. Our library was designed based on a consensus approach in which the randomization scheme reflects the frequencies of amino acids naturally occurring in respective positions responsible for antigen recognition. We demonstrate general applicability of the scaffold by selecting dVLRs specific for lysozyme and S100A7 protein with KD values in the micromolar range. The dVLR library could be used as a convenient alternative to antibodies for effective isolation of high affinity binders.

L-C1qDC-1, a novel C1q domain-containing protein from Lethenteron camtschaticum that is involved in the immune response

The C1q domain-containing (C1qDC) proteins are a family of proteins characterized by a globular C1q (gC1q) domain at their C-terminus. These proteins are involved in various processes in vertebrates and are assumed to serve as important pattern recognition receptors in innate immunity in invertebrates. Here, a novel C1qDC protein from Lethenteron camtschaticum was identified and characterized (designated as L-C1qDC-1). After a partial cDNA sequence of L-C1qDC-1 was identified in a L. camtschaticum liver cDNA library, the full-length cDNA was obtained using 3′- and 5′-rapid amplification of cDNA ends (RACE). L-C1qDC-1 encodes 236 amino acids and contains a signal peptide, a collagen-like sequence with Gly-Xaa-Yaa repeats, and a C-terminal gC1q domain. The L-C1qDC-1 protein was primarily distributed in the gut, liver and supraneural body of L. camtschaticum and was also marginally detectable in leukocytes via real-time PCR and immunofluorescence assays. Furthermore, both immunoprecipitation and immunofluorescence results showed that in L. camtschaticum serum, L-C1qDC-1 could interact with variable lymphocyte receptor (VLR) B and displayed strong colocalization with cancer cell immune responses. These results indicated that the L-C1qDC-1 gene encodes a novel C1qDC protein that may play an important role in the immune responses of L. camtschaticum, providing clues for understanding the universal functions of C1qDC proteins in other species and suggesting that these proteins could serve as pattern recognition molecules in immunotherapy.

Lamprey VLRB response to influenza virus supports universal rules of immunogenicity and antigenicity.

Immunoglobulins (Igs) are a crown jewel of jawed vertebrate evolution. Through recombination and mutation of small numbers of genes, Igs can specifically recognize a vast variety of natural and man-made organic molecules. Jawless vertebrates evolved a parallel system of humoral immunity, which recognizes antigens not with Ig, but with a structurally unrelated receptor called the variable lymphocyte receptor B (VLRB). We exploited the convergent evolution of Ig and VLRB antibodies (Abs) to investigate if intrinsic chemical features of foreign proteins determine their antigenicity and immunogenicity. Surprisingly, we find lamprey VLRB and mouse Ig responses to influenza A virus are extremely similar. Each focuses ~80% of the response on hemagglutinin (HA), mainly through recognition of the major antigenic sites in the HA globular head domain. Our findings predict basic conservation of Ab responses to protein antigens, strongly supporting the use of animal models for understanding human Ab responses to viruses and protein immunogens.

EVENTS

Jawless vertebrates have an alternative adaptive immune system mediated by variable lymphocyte receptors (VLRs), VLRA, VLRC and VLRB. In investigation on the adaptive immunity of hagfish, avian influenza virus hemagglutinin (H9N2-HA1) was used as a model antigen, with mRNA expression levels of VLRA, VLRC and Ikaros were up-regulated in the first week post-immunization. CD45 was up-regulated after the first week; and expression of VLRB progressively increased over the course of the trial. The transcriptional/translational activation of VLRB in blood was verified. The VLRBs cloned from these transcripts showed diversity in their leucine-rich repeats (LRRs). The production of specific VLRB increased in a time- and dose-dependent manner, detected by an anti-VLRB antibody (11G5). The plasma VLRB could distinguish H9N2-HA1 from unrelated proteins, but not from other HA1 subtypes. Together, our findings show that VLRs play a major role in the alternative adaptive immune system of hagfish by responding to specific foreign substances, such as H9N2-HA1.

EVENTS

Jawless vertebrates have an alternative adaptive immune system mediated by variable lymphocyte receptors (VLRs), VLRA, VLRC and VLRB. In investigation on the adaptive immunity of hagfish, avian influenza virus hemagglutinin (H9N2-HA1) was used as a model antigen, with mRNA expression levels of VLRA, VLRC and Ikaros were up-regulated in the first week post-immunization. CD45 was up-regulated after the first week; and expression of VLRB progressively increased over the course of the trial. The transcriptional/translational activation of VLRB in blood was verified. The VLRBs cloned from these transcripts showed diversity in their leucine-rich repeats (LRRs). The production of specific VLRB increased in a time- and dose-dependent manner, detected by an anti-VLRB antibody (11G5). The plasma VLRB could distinguish H9N2-HA1 from unrelated proteins, but not from other HA1 subtypes. Together, our findings show that VLRs play a major role in the alternative adaptive immune system of hagfish by responding to specific foreign substances, such as H9N2-HA1.

Evolution of vertebrate adaptive immunity: immune cells and tissues, and AID/APOBEC cytidine deaminases.

All surviving jawed vertebrate representatives achieve diversity in immunoglobulin-based B and T cell receptors for antigen recognition through recombinatorial rearrangement of V(D)J segments. However, the extant jawless vertebrates, lampreys and hagfish, instead generate three types of variable lymphocyte receptors (VLRs) through a template-mediated combinatorial assembly of different leucine-rich repeat (LRR) sequences. The clonally diverse VLRB receptors are expressed by B-like lymphocytes, while the VLRA and VLRC receptors are expressed by lymphocyte lineages that resemble αβ and γδ T lymphocytes, respectively. These findings suggest that three basic types of lymphocytes, one B-like and two T-like, are an essential feature of vertebrate adaptive immunity. Around 500 million years ago, a common ancestor of jawed and jawless vertebrates evolved a genetic program for the development of prototypic lymphoid cells as a foundation for an adaptive immune system. This acquisition preceded the convergent evolution of alternative types of clonally diverse receptors for antigens in all vertebrates, as reviewed in this article.

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.

Identification, expression pattern and potential role of variable lymphocyte receptor Aj-VLRA from Apostichopus japonicus in response to bacterial challenge

The variable lymphocyte receptors (VLRs) are found in jawless vertebrates (agnathans), and specifically recognize bacteria and viruses via their leucine-rich repeats (LRRs). VLRs are believed to be adaptive immune response molecules. Echinoderms do not have adaptive immune systems; however, in the present study, a VLR cDNA named Aj-VLRA was cloned and characterized from sea cucumber, Apostichopus japonicus. The complete cDNA of Aj-VLRA was 3072 bp, including a 1995 bp open reading frame encoding 664 amino acids comprising LRR domains, a predicted transmembrane helix and an N-terminal signal peptide. As determined by quantitative real-time reverse transcription polymerase chain reaction, Aj-VLRA transcripts are ubiquitously expressed in the body wall, longitudinal muscles, intestine and respiratory tree of A. japonicus. The expression level of Aj-VLRA was upregulated after challenge with four common marine bacteria. In situ hybridization showed that the expression of Aj-VLRA was widely distributed in the four tissues, particularly in the cytoplasm of epidermal cells. Recombinantly expressed Aj-VLRA (including the LRR domains) could bind to bacteria including Micrococcus lysodeikticus (Gram+) and Vibrio anguillarum (Gram−). Collectively, the results suggested that Aj-VLRA is related to an innate immune response of A. japonicus.

Variable Lymphocyte Receptors: A Current Overview.

Jawless vertebrates represented by lampreys and hagfish mount antigen-specific immune responses using variable lymphocyte receptors. These receptors generate diversity comparable to that of T-cell and B-cell receptors by assembling multiple leucine-rich repeat modules with highly variable sequences. Although it is true that jawed and jawless vertebrates have structurally unrelated antigen receptors, their adaptive immune systems have much in common. Most notable is the conservation of lymphocyte lineages. It appears that specialized lymphocyte lineages emerged in a common vertebrate ancestor and that jawed and jawless vertebrates co-opted different antigen receptors within the context of such lymphocyte lineages.

Structure of the OsSERK2 leucine-rich repeat extracellular domain.

Somatic embryogenesis receptor kinases (SERKs) are leucine-rich repeat (LRR)-containing integral membrane receptors that are involved in the regulation of development and immune responses in plants. It has recently been shown that rice SERK2 (OsSERK2) is essential for XA21-mediated resistance to the pathogen Xanthomonas oryzae pv. oryzae. OsSERK2 is also required for the BRI1-mediated, FLS2-mediated and EFR-mediated responses to brassinosteroids, flagellin and elongation factor Tu (EF-Tu), respectively. Here, crystal structures of the LRR domains of OsSERK2 and a D128N OsSERK2 mutant, expressed as hagfish variable lymphocyte receptor (VLR) fusions, are reported. These structures suggest that the aspartate mutation does not generate any significant conformational change in the protein, but instead leads to an altered interaction with partner receptors.

Evolution of Innate Immunity: Clues from Invertebrates via Fish to Mammals.

Host responses against invading pathogens are basic physiological reactions of all living organisms. Since the appearance of the first eukaryotic cells, a series of defense mechanisms have evolved in order to secure cellular integrity, homeostasis, and survival of the host. Invertebrates, ranging from protozoans to metazoans, possess cellular receptors, which bind to foreign elements and differentiate self from non-self. This ability is in multicellular animals associated with presence of phagocytes, bearing different names (amebocytes, hemocytes, coelomocytes) in various groups including animal sponges, worms, cnidarians, mollusks, crustaceans, chelicerates, insects, and echinoderms (sea stars and urchins). Basically, these cells have a macrophage-like appearance and function and the repair and/or fight functions associated with these cells are prominent even at the earliest evolutionary stage. The cells possess pathogen recognition receptors recognizing pathogen-associated molecular patterns, which are well-conserved molecular structures expressed by various pathogens (virus, bacteria, fungi, protozoans, helminths). Scavenger receptors, Toll-like receptors, and Nod-like receptors (NLRs) are prominent representatives within this group of host receptors. Following receptor–ligand binding, signal transduction initiates a complex cascade of cellular reactions, which lead to production of one or more of a wide array of effector molecules. Cytokines take part in this orchestration of responses even in lower invertebrates, which eventually may result in elimination or inactivation of the intruder. Important innate effector molecules are oxygen and nitrogen species, antimicrobial peptides, lectins, fibrinogen-related peptides, leucine rich repeats (LRRs), pentraxins, and complement-related proteins. Echinoderms represent the most developed invertebrates and the bridge leading to the primitive chordates, cephalochordates, and urochordates, in which many autologous genes and functions from their ancestors can be found. They exhibit numerous variants of innate recognition and effector molecules, which allow fast and innate responses toward diverse pathogens despite lack of adaptive responses. The primitive vertebrates (agnathans also termed jawless fish) were the first to supplement innate responses with adaptive elements. Thus hagfish and lampreys use LRRs as variable lymphocyte receptors, whereas higher vertebrates [cartilaginous and bony fishes (jawed fish), amphibians, reptiles, birds, and mammals] developed the major histocompatibility complex, T-cell receptors, and B-cell receptors (immunoglobulins) as additional adaptive weaponry to assist innate responses. Extensive cytokine networks are recognized in fish, but related signal molecules can be traced among invertebrates. The high specificity, antibody maturation, immunological memory, and secondary responses of adaptive immunity were so successful that it allowed higher vertebrates to reduce the number of variants of the innate molecules originating from both invertebrates and lower vertebrates. Nonetheless, vertebrates combine the two arms in an intricate inter-dependent network. Organisms at all developmental stages have, in order to survive, applied available genes and functions of which some may have been lost or may have changed function through evolution. The molecular mechanisms involved in evolution of immune molecules, might apart from simple base substitutions be as diverse as gene duplication, deletions, alternative splicing, gene recombination, domain shuffling, retrotransposition, and gene conversion. Further, variable regulation of gene expression may have played a role.

Selection of the lamprey VLRC antigen receptor repertoire.

The alternative adaptive immune system of jawless vertebrates is based on different isotypes of variable lymphocyte receptors (VLRs) that are composed of leucine-rich repeats (LRRs) and expressed by distinct B- and T-like lymphocyte lineages. VLRB is expressed by B-like cells, whereas VLRA and VLRC are expressed by two T-like lineages that develop in the thymoid, a thymus-like structure in lamprey larvae. In each case, stepwise combinatorial insertions of different types of short donor LRR cassettes into incomplete germ-line genes are required to generate functional VLR gene assemblies. It is unknown, however, whether the diverse repertoires of VLRs that are expressed by peripheral blood lymphocytes are shaped by selection after their assembly. Here, we identify signatures of selection in the peripheral repertoire of VLRC antigen receptors that are clonally expressed by one of the T-like cell types in lampreys. Selection strongly favors VLRC molecules containing four internal variable leucine-rich repeat (LRRV) modules, although VLRC assemblies encoding five internal modules are initially equally frequent. In addition to the length selection, VLRC molecules in VLRC(+) peripheral lymphocytes exhibit a distinct pattern of high entropy sites in the N-terminal LRR1 module, which is inserted next to the germ-line-encoded LRRNT module. This is evident in comparisons to VLRC gene assemblies found in the thymoid and to VLRC gene assemblies found in some VLRA(+) cells. Our findings are the first indication to our knowledge that selection operates on a VLR repertoire and provide a framework to establish the mechanism by which this selection occurs during development of the VLRC(+) lymphocyte lineage.

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

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.

Cellular and molecular characterization of hagfish VLR-based adaptive immune system (VET2P.1043)

Abstract Jawless vertebrates have an alternative adaptive immune system in which variable lymphocyte receptors (VLR) are somatically diversified through recombinatorial assembly of leucine-rich repeat cassettes during lymphocyte development. Three VLR loci (VLRA, VLRB, and VLRC) have been defined in lampreys and each is expressed by a distinct lymphocyte population. Lamprey VLRA and VLRC are expressed by T-like lineages of lymphocytes, whereas VLRB is expressed by a B-like lineage of lymphocytes, much like αβ T, γδ T and B cells in jawed vertebrates. Recently we have revised the nomenclature for the hagfish VLRs by defining a third VLR gene. Here, monoclonal and polyclonal antibodies were generated against invariant residues of hagfish VLRA, VLRB and VLRC and these were used to identify VLR-bearing lymphocytes and their products. We found that hagfish VLRA, VLRB and VLRC are expressed by three separate lymphocyte populations. Size-exclusion chromatography and western blot analysis indicated that multivalent VLRB proteins are released into the circulation, which suggests that hagfish VLRB+ cells are B-cell like. In contrast, neither VLRA nor VLRC was detectable in the plasma, supporting the hypothesis that these two lymphocyte populations are T-cell like. The demonstration of three lymphocyte populations in both lampreys and hagfish suggests that this tripartite lymphocytic differentiation program already existed in a common ancestor of the two cyclostome lineages around 480 Mya.

Copy number and sequence variation of leucine-rich repeat modules suggests distinct functional constraints operating on variable lymphocyte receptors expressed by agnathan T cell-like and B cell-like lymphocytes.

Unlike jawed vertebrates that use T cell and B cell receptors for antigen recognition, jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLR) as antigen receptors. VLRs generate high levels of diversity by assembling variable leucine-rich repeat (LRR) modules. Of the three VLRs thus far identified, VLRB is expressed on B cell-like lymphocytes and functions as antibodies, whereas VLRA and VLRC are expressed on T cell-like lymphocytes and function as membrane-bound receptors. In the present study, we show that the copy number of LRRV modules in lamprey and hagfish VLRB transcripts follows a binominal distribution with the success rates of 15.5 and 22.4 %, respectively. By contrast, the copy number distribution of LRRV modules in VLRA and VLRC transcripts deviates from the binominal distribution mainly because transcripts with two or less LRRV modules occur infrequently. Notably, the second LRRV module shows distinctive sequence signatures in VLRA and VLRC, but not in VLRB transcripts. These observations suggest that distinct functional constraints operate on VLRs expressed by agnathan T cell-like and B cell-like lymphocytes.

Negative Selection of Self-Reactive Chicken B Cells Requires B Cell Receptor Signaling and Is Independent of the Bursal Microenvironment

Although the negative selection of self-reactive B cells in the bone marrow of mammals has been clearly demonstrated, it remains unclear in models of gut-associated B cell lymphopoiesis, such as that of the chicken (Gallus gallus). We have generated chicken surface IgM-related receptors in which the diversity region of the lamprey variable lymphocyte receptor (VLR) has been fused to the C region of chicken surface IgM (Tμ). Expression of a VLR:Tμ receptor with specificity for PE supported normal development of B cells, whereas a VLR:Tμ receptor specific to hen egg lysozyme (a self-antigen with respect to chicken B cells) induced, in vivo, complete deletion of VLR(HEL)Tμ-expressing B cells. In ovo i.v. injection of PE resulted in deletion of VLR(PE)Tμ-expressing Β cells in the embryo spleen, demonstrating that negative selection was independent of the bursal microenvironment. Although chickens transduced with a murine CD8α:chicken Igα fusion protein contained B cells expressing mCD8α:chIgα, cotransfection of the mCD8α:chIgα construct, together with thymus leukemia Ag (a natural ligand for mCD8α), resulted in reduced levels of mCD8α:chIgα-expressing B cells in inverse proportion to the levels of thymus leukemia Ag-expressing cells. Deletion of mCD8α:chIgα-expressing cells was specific for B cells and required active signaling downstream of the mCD8α:chIgα receptor. Ag-mediated negative selection of developing chicken B cells can therefore occur independently of the bursal microenvironment and is dependent on signaling downstream of the BCR.

Ligation of Surface Ig by Gut-Derived Antigen Positively Selects Chicken Bursal and Peripheral B Cells

In many mammals and birds, B cell lymphopoiesis takes place in GALT, such as the avian bursa of Fabricius. Although BCR expression is sufficient for bursal colonization, the role of BCR ligation in the later stages of bursal B cell lymphopoiesis remains elusive. To address this directly, we introduced a surface Ig-related construct with defined Ag specificity containing the Ag-binding portion of a lamprey variable lymphocyte receptor specific for PE fused to a truncated chicken μ-chain (VLR(PE)Tμ) into developing chick embryos. VLR(PE)Tμ expression supports bursal follicle colonization, clonal expansion, and Ig V gene diversification. VLR(PE)Tμ-expressing B cells migrate to the periphery in the absence of the Ag starting from day 18 of embryogenesis. VLR(PE)Tμ-expressing B cells declined rapidly in the bursa and periphery in the absence of Ag after hatch; however, intrabursal injection of PE prolonged survival of VLR(PE)Tμ(+) bursal and peripheral B cells. Intrabursal introduction of Ag increased emigration of short-lived LT2(+) B cells. Peripheral VLR(PE)Tμ(+) B cells were maintained following intrabursal PE application and contained both short-lived LT2(+) and long-lived LT2(-) B cells. In the chicken bursa, the later stages of B cell development occur in the presence of gut-derived Ag; therefore, we conclude that Ag-mediated ligation of BCR in bursal B cells acts to positively select bursal B cells into both short-lived and long-lived peripheral B cell populations.

Crystal structure of the lamprey variable lymphocyte receptor C reveals an unusual feature in its N-terminal capping module.

Jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLRs) as antigen receptors to mount adaptive immune responses. VLRs generate diversity that is comparable to immunoglobulins and T-cell receptors by a gene conversion-like mechanism, which is mediated by cytosine deaminases. Currently, three types of VLRs, VLRA, VLRB, and VLRC, have been identified in lampreys. Crystal structures of VLRA and VLRB in complex with antigens have been reported recently, but no structural information is available for VLRC. Here, we present the first crystal structure of VLRC from the Japanese lamprey (Lethenteron japonicum). Similar to VLRA and VLRB, VLRC forms a typical horseshoe-like solenoid structure with a variable concave surface. Strikingly, its N-terminal cap has a long loop with limited sequence variability that protrudes toward the concave surface, which is the putative antigen-binding surface. Furthermore, as predicted previously, its C-terminal cap lacks a highly variable protruding loop that plays an important role in antigen recognition by lamprey VLRA and VLRB. Recent work suggests that VLRC+ lymphocytes in jawless vertebrates might be akin to γδ T cells in jawed vertebrates. Structural features of lamprey VLRC described here suggest that it may recognize antigens in a unique manner.

Evolution of adaptive immunity: implications of a third lymphocyte lineage in lampreys.

An alternative antigen receptor, named the variable lymphocyte receptor (VLR), was first identified in lampreys in 2004. Since then, the mechanism of VLR diversification via somatic gene assembly and the function of VLR-expressing lymphocytes have been the subject of much research. VLRs comprise leucine-rich repeat (LRR) motifs and are found only in the most phylogenetically distant vertebrates from mammals, lampreys, and hagfish. Previous reports showed that VLRA and VLRB are reciprocally expressed by lymphocytes that resemble T- and B cells; however, more recent reports show that another VLR, VLRC, is expressed on a third lymphocyte lineage, which may be equivalent to γδ T cells. The existence of three major lymphocyte lineages - one B-cell-like and two T-cell-like - and their development in lampreys, parallels the mammalian adaptive immune system. This suggests that these three cell lineages were present in the common vertebrate ancestor approximately 500 million years ago.