A Convergent Immunological Holy Trinity of Adaptive Immunity in Lampreys: Discovery of the Variable Lymphocyte Receptors

Beginning in the 1960s, there were two major schools of comparative immunology, one at the University of Minnesota managed by Robert Good and the other in Los Angeles (University of California, Los Angeles) directed by William Hildemann. Robert Good was a pioneer in the general field of cellular immunology and reported some of the first studies on T and B cells, stemming from his laboratory’s work on the thymus and bursa. He also was renowned for his studies of pediatric SCID (3). Hildemann, first a transplantation biologist, became totally dedicated to the study of comparative immunology, both in vertebrates and invertebrates (4). Although, as mentioned, adaptive responses and bona fide Abs were detected in all jawed vertebrates, for the jawless fish, Good’s group (5, 6) demonstrated AIS in lamprey but not hagfish. Hildemann (7–9), in contrast, could detect hagfish adaptive responses and attributed Good’s failure perhaps to poor animal husbandry for this species. By the early 1970s, most adherents to the field felt that AIS was operable for both cellular and humoral reactions in agnathans. Indeed, a molecule thought to be hagfish Ig was identified in Hildemann’s (10) laboratory; by contrast, the inducible Ag-specific serum molecule in lampreys discovered in Good’s laboratory was not so Ig-like, at best a putative multimer of unusual Ig heavy chains (11).

The field arrived at stasis over the next few decades, gradually teetering into an abyss. Work on the hagfish Ig was at first promising, with data suggesting that it had an unusual, perhaps primordial structure (12, 13). However, further study showed it not to be an Ig at all, but fragments of the complement component C3 (14, 15). Many studies using potential cross-reactive antisera to Ig/TCR/MHC, mammalian gene probes for cross-hybridization, or PCR with degenerate primers, among other procedures, yielded failure after failure. In a special-featured issue in Immunological Reviews on comparative immunology in 1998, all of our documented and unpublished failures were acknowledged, leading to a general feeling that there was no adaptive immunity in the agnathans [i.e., the early AIS studies mentioned above were either flawed or overinterpreted (reviewed in Ref. 16)]. Yet, hope nevertheless sprang eternal that the experiments from the 1960s would eventually be understood/redeemed and the molecules underlying agnathan adaptive immunity would be uncovered, be they the Immunological Holy Trinity or not.

In 2002, the first expressed-sequence tag project of potential lamprey lymphocytes was performed by Jan Klein and Robert Good’s protégé Max Cooper (17, 18), who is credited with the discovery of B cells in chickens (19, 20). Lamprey blood cells with the physical properties of lymphocytes were identified via their FACS light-scatter properties. Sadly, this resulting database likewise failed to expose Ig, TCR, or MHC (including class I, class II, immunoproteasome, TAP, TAPASIN, etc.) but did yield many genes homologous to lymphocyte-expressed genes in mammals, including cell-surface molecules, transcription factors, and cytokines/chemokines. Additionally, nonrearranging genes encoding Ig superfamily domains related to gnathostome Ig/TCR/MHC and CD4 were found, potentially related to their precursors (21). These studies, although heroic at the time, did not afford much solace to our community, and we were trapped in Dante’s Fifth Circle of Hell, moving down five circles from Limbo. There had to be another way to attack this problem.

In 2002, Zeev Pancer visited my laboratory in Baltimore, asking my advice on potential laboratories in the USA where he might ply his trade. By that time, Zeev was one of the most experienced comparative immunologists in the world, having worked in three different laboratories on sponge, tunicate, and sea urchin immunology. He became an absolutely fearless molecular biologist, in his last stint in Eric Davidson’s laboratory at California Institute of Technology analyzing a very large family of scavenger receptors (22), which became a model in sea urchins for other immune families such as TLR and NLR (23). I suspected that Cooper’s laboratory at the University of Alabama at Birmingham (UAB) might be in need of an excellent molecular immunologist to further scrutinize the new lamprey transcriptome databases as well as develop new ways to study this nagging dilemma of lamprey immunity at the cellular and molecular levels. Zeev was accepted into Max’s laboratory (Fig. 1) and immediately tackled the problem, publishing the first paper on the lamprey Ig superfamily genes (21).

In the classic paper highlighted in this piece (24, 25), Zeev’s new strategy was not to search for known genes but rather to determine, in an unbiased way, what molecules would be used by the lamprey for their adaptive responses. Thus, Zeev immunized larval lamprey simultaneously with multiple immunostimulants, including bacteria, sheep RBCs, PHA, and PWM (the proverbial kitchen sink), and then examined their responses. By FACS, there was a large population of lymphoblasts detected by light scatter after immunization, demonstrating that the procedure had indeed resulted in cell activation. RNA prepared from these cells resulted in high levels of transcripts encoding multiple leucine-rich repeat (LRR) cassettes, and cDNAs encoding these domains could be grouped into many subsets with the same N- and C-terminal LRR cassettes but with varying numbers of internal LRR cassettes; even the subsets with the same number of cassettes were diverse in sequence. Thus, there were either many germline genes in this family, similar to what was found in sea urchins for their innate immune gene families (23), or a somatic mechanism generated the VLR repertoire. Germline DNA showed that the N- and C-terminal cassettes were encoded at a single locus, and short exons encoding the internal LRRs in cDNAs were found 5′ and 3′ of this central locus. PCR amplicons from blood cell–genomic DNA showed that some of the flanking LRR cassettes were stitched to each other between the invariant LRR cassettes (Fig. 1). Like in gnathostome lymphocytes, the lamprey cells expressed one VLR per cell (i.e., were allelically excluded). No recombination signal sequences were detected at the borders of the LRR, suggesting that RAG1/2 was not involved in the rearrangement events. The take-home message from this first paper, and it was indeed a stunner, was that there were Ag receptors expressed by lamprey lymphocytes convergent with Ig/TCR, the so-called variable lymphocyte receptors or VLR, which were generated by a non–RAG-based rearrangement events (25). Mature lymphocytes stimulated by Ag then secreted the VLR into the plasma (Fig. 1).

For run-of-the-mill immunologists, this paper reported one of the most unexpected and exciting discoveries in modern immunology. We, the comparative immunologists, were equally flabbergasted, but on top of that, we breathed a collective sigh of relief that this old and contentious problem had been solved.

Like many other instances in biology, after a seminal discovery is made, it is followed up with one interesting result after another (reviewed in Ref. 26). Cooper’s (27) UAB group had been excellent at producing mAbs to many cell-surface receptors over the years, and they generated VLR-specific reagents lickety-split. Adaptive responses were demonstrated to several foreign Ags, and VLR-positive cells were followed in lamprey larvae in vivo. The secreted VLR form pentamers of dimers, reminiscent of IgMs of all gnathostomes (also by convergence), and these were visualized first by electron microscopy (28), and later individual VLR structures were revealed by crystallography (29–31) [note that the VLR are likely the induced molecules to carbohydrate Ags found in 1970 (11)]. Soon, the VLR were also found in hagfish (32). Pancer moved to the Center of Marine Biotechnology in Baltimore as a principal investigator, where he showed that the diversity of the VLR was very high (like Ig/TCR) and he also detected two apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family members expressed by lymphocytes (CDA1 and CDA2) that were implicated in VLR generation of diversity (33). Sakano [codiscoverer of recombination signal sequences with Tonegawa (34) in 1979] and colleagues published data suggesting that VLR diversity is generated by a gene conversion-like process of “copy choice,” in which the LRR cassettes are brought together via homologous joining events, most likely orchestrated by the lamprey APOBEC family members (35).

A simple model for this adaptive system was that, similar to T-independent B cell responses in gnathostomes, Ag bound to cell-surface VLR along with pattern recognition receptor signals would stimulate Ag-specific naive lymphocytes to begin to secrete “antibodies” (27). That model was modified drastically with the discovery of a second VLR locus, VLRA, shown to be expressed on a subset of lymphocytes distinct from VLRB and, like TCRs, not secreted after lymphocyte stimulation (36). The transcriptomes of VLRA- and VLRB-bearing lymphocytes were similar to gnathostome T and B cells, respectively, and the two subsets seemed to use different APOBEC family members for generating and, perhaps, diversifying the repertoire. Later, a third locus, VLRC, was discovered and the cells bearing this receptor seem to have a transcriptome and tissue distribution similar to γδ T cells (37–39). Another totally unexpected finding is that the agnathans have a thymus-like structure, called the thymoid, in the pharyngeal region where VLRA and VLRC cells develop (40, 41). Previously, it was (almost) unambiguous in our field that agnathans lacked a thymus (42); exposure of the thymoid was made possible by the discovery of the VLRA and CDA1 [as well as thymus-specific transcription factors (43)], which allowed for detection of the developing cells. In summary, the follow-up discoveries to the Pancer and Cooper paper (25) in 2004 were head-turning on par with Linda Blair in The Exorcist, each new paper offering a novel and unexpected glimpse of the agnathan and, likely primordial, AIS. It is worth re-emphasizing that although the Ag receptors of jawed and jawless fish arose via convergent evolution, the lymphocyte lineages, the thymoid/thymus, and emergence of the APOBEC family occurred via divergence from a jawless/jawed common ancestor (26, 44); indeed, for the APOBEC family, there is recent evidence for its presence even in the invertebrates (45, 46).

There is general agreement now that the transcriptional control of lymphocyte development emerged in the common ancestor of jawless and jawed vertebrates, but different sets of Ag receptors were co-opted in the two lineages. The jawless fish clearly have two-thirds of a convergent Immunological Holy Trinity, Ig (VLRB) and TCR (VLRA), but the third divinity, the MHC, has proven elusive. Some evidence from the VLRA and VLRC sequences suggest that there is a conserved region in the binding sites of the diverse repertoire (47, 48), perhaps for some form of positive selection and later Ag recognition, but whether lamprey T cells recognize peptides, or instead a form of cell-bound native Ag (31), is totally unknown. If I were to wager, I’d bet on a convergent MHC, a cell-surface molecule [perhaps like an Fc or complement receptor or a polymorphic receptor (49, 50)] on APC that displays native Ag. But I wouldn’t bet the house.

The emergence of an organ for differentiation of T cells is also intriguing. I always thought the driving force for thymic emergence was to dedicate a specialized organ for T cell–positive selection, but perhaps there is a simpler explanation: a dedicated organ was required to sequester T cell development away from B cell development (51). If B cell responses require T cell help in agnathans, where does this happen? Unlike the gnathostomes, which all have a spleen (unless it was lost), no dedicated secondary lymphoid tissue has been detected in agnathans (52). Thus, the compartmentalization of adaptive immunity and Ag presentation to T cells and B cells, including identification of the APC themselves, have not been addressed. So many other questions arise, including the following, to name a few. Are there T cell subsets (CD4 and CD8, as well as T-helper phenotypes and what is the cytokine repertoire)? Do innate subsets of T and B cells exist as well as innate lymphoid cells (53)? How does the transition from cell-surface GPI-linked VLRB to secreted pentameric Ab occur? What are the signaling requirements for lymphocyte activation? Is there a form of affinity maturation in immune responses (54)? Are there striking differences in the AISs of larval versus adult lamprey, similar to what is seen in amphibians (55)? Is memory operable in the agnathan AIS? Is there any evidence for AIS in animals whose ancestors predated the vertebrates (43, 56)? We should not forget the translational potential of the VLR (57) as well as the use of the lamprey APOBEC family members in mutagenesis studies (58). With the emergence of the CRISPR/Cas technology and cell-culture techniques in lampreys (59), all of these problems will be addressed in the coming years. For so many scientific, psychological, and spiritual reasons, we owe a great debt to Pancer, Cooper, and their colleagues for their discovery of these convergent Ag receptors (25).

I thank Louis Du Pasquier and Mike Criscitiello for critical reading and Nick Cohen and the late Bill Clem for discussions of the history of agnathan immune studies.

The author has no financial conflicts of interest.