Immunology

Another manifestation of GOD

In studies of the evolution of the adaptive immune system, the lamprey has been an unlikely centre of attention. These studies now provide evidence of a fascinating variation on how such a system can operate.

Like almost all other vertebrates, we humans have jaws. Deep in vertebrate family history, however, are our jawless relatives, most of which are extinct. The exception is the lamprey, which, along with its cousin the hagfish, is alive and well, and has posed a real puzzle for immunologists: lampreys seem to get by without one of the two arms of jawed-vertebrate immune defence, the so-called adaptive system, which is well developed in the oldest jawed vertebrates, the sharks. As Pancer et al. show on page 174 of this issue1, however, lampreys apparently have such a system. It's just rather different.

The other arm of the immune system is the innate system, which reacts swiftly to invaders with a generalized response mounted by receptors encoded in the germ line. The more glamorous adaptive system is defined by non-germline-generated receptors that bind an invader's signature molecules, or antigens, and can recognize any biological molecule in nature. These antigen receptors — antibodies or T-cell receptors — are generated by a gene-rearrangement process during lymphocyte development2. As shown in Fig. 1a, the antigen-binding or variable (V) region is formed by the rearrangement of V, D (diversity) and J (joining) gene segments for one chain of the receptor, and of V and J segments for the other chain (for antibodies' heavy and light chains, respectively). The enzymes RAG1 and RAG2 stitch these segments together3. The multigenic nature and imprecise joining of the V, D and J segments result in each individual having a huge repertoire of antigen receptors.

Figure 1: Adaptive immunity in jawed vertebrates and the jawless lamprey.
figure1

a, Antibody production as the example for jawed vertebrates. During lymphocyte development, the RAG enzymes rearrange genes by recognizing recombination signal sequences (RSS) that flank the variable (V), diversity (D) and joining (J) gene segments at heavy-chain and light-chain loci (C, constant regions). These segments are stitched together by enzymes, and the functional gene encodes the binding sites that can recognize foreign antigen. A huge variety of such sites can be produced. But only one type of antibody is expressed by each lymphocyte; thus, only cells expressing antibodies specific for a particular antigen will become stimulated, proliferate by clonal selection and secrete antibodies to destroy an invader. b, Variable lymphocyte receptors (VLRs) in the lamprey1. The VLR genetic locus consists of segments at each end of the functional gene that encode leucine-rich repeats (NLRR or CLRR). By an unknown mechanism, other LRR gene cassettes from upstream and downstream of these segments are inserted to produce a functional gene. Preliminary indications are that each lamprey lymphocyte has a specific VLR, implying that clonal selection also operates in this system. VLRs are probably anchored to the lymphocyte membrane and released after antigenic stimulation.

Immunologists irreverently refer to this process as GOD (generation of diversity). For such a system to be efficient, the antigen receptors must be expressed in clones of lymphocytes4 — B cells for antibodies and T cells for T-cell receptors. Only those lymphocytes that can bind to a particular antigen are stimulated to secrete anti-pathogen antibodies (B cells) or produce ‘effector’ T cells that can, directly or indirectly, kill our own cells infected by intra-cellular invaders.

Evidence for the existence of this adaptive system in invertebrates and in lampreys and hagfish has been pursued valiantly, but in vain. The failed search for antibodies and T-cell receptors — as well as for other components of the adaptive system, such as genes encoding the major histocompatibility complex (MHC) important for T-cell stimulation, the tissues where the lymphocytes are generated and then stimulated by antigen, and the RAG genes themselves — has led to the premature end of several graduate student careers. We in the comparative immunology field now think it unlikely that such apparatus will be uncovered outside the jawed vertebrates5. The unusual nature of the RAG genes, and the recombination signal sequences that they recognize (Fig. 1a), have led to a ‘big bang’ hypothesis6, in which mobile genetic elements, including the RAG genes, fortuitously invaded and disrupted an ancestral antibody–T-cell-encoding genomic sequence7. These sequences could then be stitched together only by RAG action, resulting in a mechanism for GOD and the conception of the jawed-vertebrate adaptive immune system.

Now wait one minute — all this does not mean that GOD could not be realized in another way in jawless vertebrates or invertebrates. Indeed, the same end can be achieved by mutational or gene-conversion processes in some jawed vertebrates, and such mechanisms could have preceded the RAG invasion8. Furthermore, GOD in jawed vertebrates is not the only prototype for gene rearrangement in the animal kingdom: parasites have long been known to have mobile genes that create rapidly evolving proteins capable of tricking the host's immune system9.

Rather than assuming that if lampreys had an adaptive immune system it would (perhaps) be a forerunner of the vertebrate system, Pancer et al.1 wondered whether any adaptive response would be carried out by lymphocytes. So they performed a search for lymphocyte-specific genes. In earlier work in collaboration with another laboratory, this group purified lamprey cells on the basis of their lymphocyte-like properties and searched for expressed sequence tags (ESTs) within these cells10. Like the rest of us, they found no antibodies, T-cell receptors, MHC and so on. But they did unearth ESTs for transcription factors, cell-surface molecules and other proteins that are expressed more or less specifically in jawed-vertebrate lymphocytes. They also found unique ESTs for leucine-rich repeats (LRRs), which are typical of many proteins involved in immune defence11.

Pancer et al.1 have now shown that these genes are upregulated in antigen-stimulated lymphocytes. Additionally, they found that this gene family is extremely diverse in the number and types of LRR ‘cassettes’ encoded by each complementary DNA clone, and therefore dubbed them ‘variable lymphocyte receptors’ or VLRs. This diversity could have been the result of either a vast number of germline genes, as for other innate receptors12, or of non-germline rearrangement. Pancer et al. found that invariant LRR cassettes were encoded by each end of each clone of complementary DNA, but that LRRs sandwiched between them varied in number (Fig. 1b). Inspection of genomic DNA showed the invariant cassettes to be encoded by single-copy genes near to each other in non-lymphocyte DNA. In further analyses of lymphocyte genomic DNA, it emerged that the other LRR cassettes were somehow added and sewn together. Furthermore, preliminary evidence suggests that each lamprey lymphocyte bears a unique VLR, setting the stage for clonal selection reminiscent of the RAG-based system.

This provocative discovery raises many stirring questions. How is GOD accomplished for the VLR? The LRR gene cassettes are not flanked by signal sequences recognized by RAG proteins, so the VLR diversity-generating mechanism has probably arrived at the same general principle as the antibody–T-cell-receptor system from different origins. Can the VLR account for old experiments that suggested adaptive immunity in jawless fish5? This is possible, but because the VLR are lipid-anchored to the lymphocytes they would have to be clipped off to be secreted into body fluids and bind to foreign invaders, a totally different mechanism from that for the initially transmembrane antibodies in jawed vertebrates.

Is this an old system that was superseded by jawed-vertebrate immunity, or is it something that arose only in a lamprey ancestor? The evolutionary split between lampreys and hagfish occurred more than 300 million years ago; if hagfish (and perhaps some invertebrates or even jawed vertebrates as well) have VLRs, it suggests that the ‘rules’ for the generation of an adaptive immune system arose early in evolution, and indeed that the VLR system was superseded by our current adaptive system. If VLRs are the foundation of the lamprey adaptive immune system, do the lymphocytes expressing them form a single subset? Or, as in the jawed vertebrates, are there subsets of cells for different functions?

Revelations of other immune diversity-generating mechanisms may be on the way. Data from invertebrates such as echinoderms and molluscs (L. C. Smith, George Washington Univ.; E. Loker, Univ. New Mexico, personal communications) suggest that non-germline mechanisms for GOD might abound in the animal kingdom, especially among animals that are relatively long-lived or complex, or both. The jawed-vertebrate adaptive immune system, based on RAG enzymes, was a magnificent and unique innovation. But Pancer et al. have shown that the RAG-based system is not the be-all and end-all of adaptive immunity.

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Flajnik, M. Another manifestation of GOD. Nature 430, 157–158 (2004). https://doi.org/10.1038/430157a

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