T-like Lymphocytes Research Articles

Evolution of variable lymphocyte receptor B antibody loci in jawless vertebrates

SignificanceJawless vertebrates, lampreys and hagfish, use three types of variable leucine-rich repeat proteins as antigen receptors: VLRB on B-like cells and VLRA or VLRC on T-like lymphocytes. In their incomplete germline status, theVLRBgenes in different lamprey species uniquely possess long noncoding intervening regions, which contain different sets of transposable elements and potential donor cassettes. Phylogenetic analysis of the germlineVLRgenes reveals ongoing evolution over the last 250 million y. Comparative assessment of genomic sequences in two lamprey species yields a detailed map of theirVLRBloci. The use of this map, together with an analysis of cassette usage in a large repertoire of expressedVLRBs, supports a fragmented Lego-like model ofVLRBassembly.

Ancient BCMA-like Genes Herald B Cell Regulation in Lampreys.

The TNF superfamily ligands BAFF and APRIL interact with three receptors, BAFFR, BCMA, and TACI, to play discrete and crucial roles in regulating B cell selection and homeostasis in mammals. The interactions between these ligands and receptors are both specific and redundant: BAFFR binds BAFF, whereas BCMA and TACI bind to either BAFF or APRIL. In a previous phylogenetic inquiry, we identified and characterized a BAFF-like gene in lampreys, which, with hagfish, are the only extant jawless vertebrates, both of which have B-like and T-like lymphocytes. To gain insight into lymphocyte regulation in jawless vertebrates, in this study we identified two BCMA-like genes in lampreys, BCMAL1 and BCMAL2, which were found to be preferentially expressed by B-like lymphocytes. In vitro analyses indicated that the lamprey BAFF-like protein can bind to a BCMA-like receptor Ig fusion protein and to both BCMAL1- and BCMAL2-transfected cells. Discriminating regulatory roles for the two BCMA-like molecules are suggested by their differential expression before and after activation of the B-like lymphocytes in lampreys. Our composite results imply that BAFF-based mechanisms for B cell regulation evolved before the divergence of jawed and jawless vertebrates.

Origin and Evolution of Dendritic Epidermal T Cells.

Dendritic epidermal T cells (DETCs) expressing invariant Vγ5Vδ1 T-cell receptors (TCRs) play a crucial role in maintaining skin homeostasis in mice. When activated, they secrete cytokines, which recruit various immune cells to sites of infection and promote wound healing. Recently, a member of the butyrophilin family, Skint1, expressed specifically in the skin and thymus was identified as a gene required for DETC development in mice. Skint1 is a gene that arose by rodent-specific gene duplication. Consequently, a gene orthologs to mouse Skint1 exists only in rodents, indicating that Skint1-dependent DETCs are unique to rodents. However, dendritic-shaped epidermal γδ T cells with limited antigen receptor diversity appear to occur in other mammals. Even lampreys, a member of the most primitive class of vertebrates that even lacks TCRs, have γδ T-like lymphocytes that resemble DETCs. This indicates that species as divergent as mice and lampreys share the needs to have innate-like T cells at their body surface, and that the origin of DETC-like cells is as ancient as that of lymphocytes.

Evolutionary modification of the VLR-based adaptive immune system in jawless vertebrates: Functional implications

Abstract The three types of variable lymphocyte receptor genes, VLRA, VLRB and VLRC, in the extant jawless vertebrates, encode antigen receptors, the remarkably diverse repertoire of which is generated by insertion of neighboring leucine rich repeat (LRR) sequences into the incomplete germline genes. In both lampreys and hagfish, the B-cell like VLRB+ cells differentiate into VLRB-secreting plasma cells, whereas the αβ and γδ T cell-like VLRA+ and VLRC+ cells express their VLR products solely as cell surface proteins. However, in comparative studies of lampreys and hagfish, we find that remarkable functional differences have evolved in these lymphocyte lineages. In contrast with their striking predominance in lampreys, the VLRB+ cells constitute a minor lymphocyte population in hagfish, and the VLRC+ cells are predominate. Notably the germline VLRB gene in hagfish contains a short non-coding intervening sequence, whereas the VLRB genes in sea lampreys and Japanese lampreys have very long intervening sequences which contain multiple transposable elements that may influence VLRB expression. In keeping with the relative low numbers of hagfish VLRB+ cells, antibody responses to a model immunogen, sheep erythrocytes, are much less robust in hagfish than in lampreys. Thus, even though the fundamental genetic program for differentiation of two prototypic T-like lymphocyte lineages and one B-like lineage is conserved in both jawless and jawed vertebrates, the genetic programs used for fine tuning of the VLR-based immunity have undergone notable independent evolutionary changes in lampreys and hagfish over the past ~480 million years.

Evolution of Alternative Adaptive Immune Systems in Vertebrates.

Adaptive immunity in jawless fishes is based on antigen recognition by three types of variable lymphocyte receptors (VLRs) composed of variable leucine-rich repeats, which are differentially expressed by two T-like lymphocyte lineages and one B-like lymphocyte lineage. The T-like cells express either VLRAs or VLRCs of yet undefined antigen specificity, whereas the VLRB antibodies secreted by B-like cells bind proteinaceous and carbohydrate antigens. The incomplete VLR germline genes are assembled into functional units by a gene conversion-like mechanism that employs flanking variable leucine-rich repeat sequences as templates in association with lineage-specific expression of cytidine deaminases. B-like cells develop in the hematopoietic typhlosole and kidneys, whereas T-like cells develop in the thymoid, a thymus-equivalent region at the gill fold tips. Thus, the dichotomy between T-like and B-like cells and the presence of dedicated lymphopoietic tissues emerge as ancestral vertebrate features, whereas the somatic diversification of structurally distinct antigen receptor genes evolved independently in jawless and jawed vertebrates.

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.

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.

Origin and evolution of adaptive immunity.

The evolutionary emergence of vertebrates was accompanied by major morphological and functional innovations, including the development of an adaptive immune system. Vertebrate adaptive immunity is based on the clonal expression of somatically diversifying antigen receptors on lymphocytes. This is a common feature of both the jawless and jawed vertebrates , although these two groups of extant vertebrates employ structurally different types of antigen receptors and principal mechanisms for their somatic diversification . These observations suggest that the common vertebrate ancestor must have already possessed a complex immune system, including B- and T-like lymphocyte lineages and primary lymphoid organs, such as the thymus, but possibly lacked the facilities for somatic diversification of antigen receptors. Interestingly, memory formation, previously considered to be a defining feature of adaptive immunity, also occurs in the context of innate immune responses and can even be observed in unicellular organisms, attesting to the convergent evolutionary history of distinct aspects of adaptive immunity.

VLR-based adaptive immunity.

Lampreys and hagfish are primitive jawless vertebrates capable of mounting specific immune responses. Lampreys possess different types of lymphocytes, akin to T and B cells of jawed vertebrates, that clonally express somatically diversified antigen receptors termed variable lymphocyte receptors (VLRs), which are composed of tandem arrays of leucine-rich repeats. The VLRs appear to be diversified by a gene conversion mechanism involving lineage-specific cytosine deaminases. VLRA is expressed on the surface of T-like lymphocytes; B-like lymphocytes express and secrete VLRB as a multivalent protein. VLRC is expressed by a distinct lymphocyte lineage. VLRA-expressing cells appear to develop in a thymus-like tissue at the tip of gill filaments, and VLRB-expressing cells develop in hematopoietic tissues. Reciprocal expression patterns of evolutionarily conserved interleukins and chemokines possibly underlie cell-cell interactions during an immune response. The discovery of VLRs in agnathans illuminates the origins of adaptive immunity in early vertebrates.

A thymus candidate in lampreys

Immunologists and evolutionary biologists have been debating the nature of the immune system of jawless vertebrates--lampreys and hagfish--since the nineteenth century. In the past 50 years, these fish were shown to have antibody-like responses and the capacity to reject allografts but were found to lack the immunoglobulin-based adaptive immune system of jawed vertebrates. Recent work has shown that lampreys have lymphocytes that instead express somatically diversified antigen receptors that contain leucine-rich-repeats, termed variable lymphocyte receptors (VLRs), and that the type of VLR expressed is specific to the lymphocyte lineage: T-like lymphocytes express type A VLR (VLRA) genes, and B-like lymphocytes express VLRB genes. These clonally diverse anticipatory antigen receptors are assembled from incomplete genomic fragments by gene conversion, which is thought to be initiated by either of two genes encoding cytosine deaminase, cytosine deaminase 1 (CDA1) in T-like cells and CDA2 in B-like cells. It is unknown whether jawless fish, like jawed vertebrates, have dedicated primary lymphoid organs, such as the thymus, where the development and selection of lymphocytes takes place. Here we identify discrete thymus-like lympho-epithelial structures, termed thymoids, in the tips of the gill filaments and the neighbouring secondary lamellae (both within the gill basket) of lamprey larvae. Only in the thymoids was expression of the orthologue of the gene encoding forkhead box N1 (FOXN1), a marker of the thymopoietic microenvironment in jawed vertebrates, accompanied by expression of CDA1 and VLRA. This expression pattern was unaffected by immunization of lampreys or by stimulation with a T-cell mitogen. Non-functional VLRA gene assemblies were found frequently in the thymoids but not elsewhere, further implicating the thymoid as the site of development of T-like cells in lampreys. These findings suggest that the similarities underlying the dual nature of the adaptive immune systems in the two sister groups of vertebrates extend to primary lymphoid organs.

Alternative Adaptive Immunity in Jawless Vertebrates

Jawless vertebrates use variable lymphocyte receptors (VLRs) that are generated by RAG-independent combinatorial assembly of leucine-rich repeat cassettes for Ag recognition, instead of the Ig-based Ag receptors used by jawed vertebrates. The VLR genes encode for crescent-shaped proteins that use variable beta-strands and a C-terminal loop to bind to Ags rather than the six CDR loops used by BCRs and TCRs. VLR mAbs have been isolated recently, which enabled the structure of VLR-Ag complexes to be defined. The jawless vertebrate adaptive immune system has many similarities to the Ig-based system of jawed vertebrates, including the compartmentalized development of B-like and T-like lymphocyte lineages that proliferate and differentiate into VLR-secreting plasmacytes and proinflammatory cytokine-producing cells in response to Ags. The definition of common features of the VLR-based and Ig-based systems offers fresh insight into the evolution of adaptive immunity.

Proliferative responses of tilapia T-like lymphocytes to stimulation by Concanavalin A

Peripheral blood lymphocytes were cultured in vitro in the presence of a mitogen; Concanavalin A (Con A), this resulted into a process described as lymphocyte transformation. The morphological changes in the cells were studied using phase contrast and electron microscopes. The results presented corroborated the existence of lymphocyte heterogeneity in tilapias. Keywords: mitogenic stimulation, T-like lymphocytes, Concanavalin A African Journal of Biomedical Research Vol. 8(3) 2005: 151-155

Surface markers of fish T-cells

Two immunological reactions against foreign antigens can be distinguished: (1) the humoral immune response, involving the production of immunoglobulins by plasma cells; (2) the cell-mediated immune responses, mediated by thymus-derived cells. All jawed vertebrates show these two immunological pathways. In lower vertebrates, the existence of cell-mediated immunity has been shown by transplantation experiments resulting in allograft rejection. The basic phenomena observed are similar to those in mammals. Lymphocyte heterogeneity, particularly the participation of T-like lymphocytes in fish immunity, is now well established. T-like cells were defined as sIg−cells which proliferate in the presence of specific mitogens for mammalian T lymphocytes (Con A and PHA). The mixed leucocyte reaction (MLR) has been demonstrated in all vertebrates, from shark to mammals. The in vitro anti-hapten antibody response to a T-dependent antigen requires the participation of sIg+and sIg−cells and monocytes. In addition, fish sIg−lymphocytes specifically proliferate in the presence of processed and presented antigen, reminiscent of the mammalian classical T-cell/B-cell/APC collaboration. Hitherto, only a few attempts to generate antibodies, reacting exlusively with fish T-cell markers, have been successful. The use of molecular tehniques such as PCR and library screening has allowed the identification of cDNA clones encoding TCR-α and -β chains in several fish species. TCRδ-like chains not yet detected in teleosts have been isolated in shark, suggesting that TCRγδ-like receptors should exist in all gnathostomes. Whereas the teleost TCRβ locus shows a translocon organisation comparable to that of its mammalian counterpart, elasmobranch TCRβ genes are arranged in multiple clusters. As in mammals, the TCR repertoire in fish species appears to be considerable.

Phylogenesis of Lymphocyte Diversity I. Immunoglobulin Determinants on the Lymphocyte Surface of the Lizard Agama Stellio

Living lymphocytes of the adult lizard Agama stellio were examined for surface immunoglobulin (Ig) utilizing an antiserum specific for Agama γ-globulin (RAGS) raised in rabbits. In indirect immunofluorescence, 44% of bone marrow lymphocytes, 29% of splenic lymphocytes, 25% of peripheral blood lymphocytes and 7% of thymocytes exhibited readily demonstrable surface Ig determinants. Observations on shedding and resynthesis justified the endogenous production of these Ig molecules by Agama lymphocytes and ruled out false scoring due to cytophilic antibodies. Following interaction with AAGS in a temperature-controlled reaction, redistribution of the Ig-AAGS complexes into separable stages of patches, 1/2 caps, 1/4 caps, tight caps, shedding and re-expression was in a manner comparable to that cited for mammalian Ig molecules. Studies on the rate of capping and resynthesis demonstrated, however, that the processes were extremely prolonged compared to those observed in mammals and that the lymphocytes were not induced to transitional movement throughout. In terms of the ease of detectability, cross-reactivity with serum Ig, accessibility to AAGS and capping and resynthesis rates, no considerable differences in the Ig molecules of thymic and peripheral lymphocytes were observed. The Ig-bearing lymphocytes seemed to constitute a homogeneous population, which expresses selectivity in its representation within the various lymphoid compartments. The minority of Ig-bearing thymic lymphocytes was suggested to have a preferential homing capacity to the thymus environment in response to the total absence of GALT in this animal model. In structural terms, the present study suggests the possible delineation of lymphocytes into Ig-positive B-like and Ig-negative T-like lymphocytes at this evolutionary level.