Mystery Rays from Outer Space

Meddling with things mankind is not meant to understand. Also, pictures of my kids

December 31st, 2007

The Twelve Days of Mystery Rays

H-2Kb + SIEFARLI’ve saved 229 papers from 2007 in my References database. Here are some of the ones I was especially struck by this year. This doesn’t necessarily mean “most significant”, or “most dramatic”, or anything other than “I thought they were cool”.
Twelve papers — one per month1 — in alphabetical order so I don’t have to decide between them:
1. Apetoh, L., Ghiringhelli, F., Tesniere, A., Obeid, M., Ortiz, C., Criollo, A. et al. (2007). Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med, 13, 1050 – 1059.
Suggests that the real reason chemotherapy works is not that it kills the tumor cells, but rather that the tumor cells are rendered capable of priming the adaptive immune system; it’s the immune system that actually does the work of killing off the tumor. I talked about this paper here.

Meggan Gould, Crow 104
“Crow 104” by Meggan Gould

2. Brault, A. C., Huang, C. Y., Langevin, S. A., Kinney, R. M., Bowen, R. A., Ramey, W. N. et al. (2007). A single positively selected West Nile viral mutation confers increased virogenesis in American crows. Nat Genet, 39(9), 1162-1166.
An example of how a small change in a viral genome can make a huge change in its pathogenesis — this West Nile virus mutant is more lethal to crows. Read this one in conjunction with LaDeau, S. L., Kilpatrick, A. M., & Marra, P. P. (2007). West Nile virus emergence and large-scale declines of North American bird populations. Nature, 447(7145), 710-713. I talked about West Nile Virus here, though not so much about these papers.

3. Flutter, B., Fu, H. M., Wedderburn, L., & Gao, B. (2007). An extra molecule in addition to human tapasin is required for surface expression of β2m linked HLA-B4402 on murine cell. Mol Immunol, 44(14), 3528-3536.
Evidence that there is still at least one unidentified molecule that’s important for antigen presentation. Mainly interesting to me because it reflects my own paper from 2005 that shows the same thing. The description of this phenotype isn’t identical to my mutant cell line, but I suspect it’s the same molecule.

4. Garrison, K. E., Jones, R. B., Meiklejohn, D. A., Anwar, N., Ndhlovu, L. C., Chapman, J. M. et al. (2007). T Cell Responses to Human Endogenous Retroviruses in HIV-1 Infection. PLoS Pathog, 3(11), e165.
Endogenous retroviruses, the withered corpses of ancient retroviruses that litter our genome, are partially reanimated by HIV infection. The newly-expressed HERV proteins are recognized by T cells, potentially offering a non-mutating target that might enable T cells to recognize HIV-infected cells.

Colorectal cancer
Blood vessels in a colorectal cancer

5. Koebel, C. M., Vermi, W., Swann, J. B., Zerafa, N., Rodig, S. J., Old, L. J. et al. (2007). Adaptive immunity maintains occult cancer in an equilibrium state. Nature, 450(7171), 903-907.
Cancers arise from normal cells through a multi-step process; immune system evasion is one critical step in this process. It’s been proposed that nascent cancers may spend years in an undetectable state, suppressed by the immune system. Here is more or less direct evidence for this equilibrium state. I talked about this paper here and here .

6. Kotturi, M. F., Peters, B., Buendia-Laysa, F. J., Sidney, J., Oseroff, C., Botten, J. et al. (2007). The CD8+ T-cell response to lymphocytic choriomeningitis virus involves the L antigen: uncovering new tricks for an old virus. J Virol, 81(10), 4928-4940.
Mainly a technological advance in T cell epitope identification, but a dramatic example of how much there is to learn even in this very well-studied virus system. Also offers a really dramatic example of immunodominance. I talked about it here, among other places.

7. Munks, M. W., Pinto, A. K., Doom, C. M., & Hill, A. B. (2007). Viral interference with antigen presentation does not alter acute or chronic CD8 T cell immunodominance in murine cytomegalovirus infection. J Immunol, 178(11), 7235-7241.
Viruses have long been known to block the class I major histocompatibility complex antigen presentation pathway, and the presumption has been that this allows the virus to avade detection and destruction by cytotoxic T cells. But this has mainly been a presumption because the work is mainly in cultured cells, rather than in real animals. Here Ann Hill’s group continues their work looking at immune evasion molecules in authentic infections — and show that they have amazingly little impact on the infection or the immune response. I talked about it here.

8. Siddle, H. V., Kreiss, A., Eldridge, M. D., Noonan, E., Clarke, C. J., Pyecroft, S. et al. (2007). Transmission of a fatal clonal tumor by biting occurs due to depleted MHC diversity in a threatened carnivorous marsupial. Proc Natl Acad Sci U S A, 104(41), 16221-16226.
Tasmanian Devils are being killed by an infectious tumor, which apparently can spread because Devils are not very diverse at their major histocompatibility complex region — the region that usually serves to prevent graft “takes”, which is essentially what this tumor is doing. I discussed this paper here, along with the even more interesting canine transmissible venereal tumor.

9. Stemberger, C., Huster, K., M., Koffler, M., Anderl, F., Schiemann, M., Wagner, H. et al. (2007). A Single Naive CD8+ T Cell Precursor Can Develop into Diverse Effector and Memory Subsets. Immunity, 27(6), 985-997.
How many T cells are there that recognize a particular antigen, and how much expansion do they undergo to form the immune response we all know and love? I talked about a previous paper that counted T cells — here is evidence that a single cell can form multiple branches of an immune response.

Baines HSV
Electron tomogram of HSV2

10. Verjans, G. M., Hintzen, R. Q., van Dun, J. M., Poot, A., Milikan, J. C., Laman, J. D. et al. (2007). Selective retention of herpes simplex virus-specific T cells in latently infected human trigeminal ganglia. Proc Natl Acad Sci U S A, 104(9), 3496-3501.
Herpes simplex virus has traditionally been believed to enter a latent state in human trigenimal ganglia during which no viral proteins are produced. This paper is one of a series that shows that the immune system recognizes neurons latently infected with HSV, which strongly suggests that there is some very low-level protein expression, offering a mechanism by which the virus can interact with the world outside its neuron. I talked about it here.

11. Wearsch, P. A. & Cresswell, P. (2007). Selective loading of high-affinity peptides onto major histocompatibility complex class I molecules by the tapasin-ERp57 heterodimer. Nat Immunol, 8(8), 873-881.
After years of trying, here at last is efficient in vitro MHC class I peptide loading. I talked about it here.

12. Zhang, S.-Y., Jouanguy, E., Ugolini, S., Smahi, A., Elain, G., Romero, P. et al. (2007). TLR3 Deficiency in Patients with Herpes Simplex Encephalitis. Science, 317(5844), 1522-1527.
Herpes simplex encephalitis is a rare, but devastating, complication of herpes simplex infection. Here Zhang et al show that a defect in TLR3 — not generally associated with HSV — is associated with this disease. I talked about the paper here.

  1. In other words, I couldn’t narrow it down to the traditional ten.[]
  2. Electron tomogram of a HSV nucelocapsid completing envelopment, from Baines, J. D., C. E. Hsieh, E. Wills, C. Mannella, and M. Marko. 2007. Electron tomography of nascent herpes simplex virus virions. J Virol 81: 2726-2735.[]
December 26th, 2007

Any excuse to say “axolotl”

Axolotl My last post talked about various species that have limited MHC diversity in their population. I got carried away and forgot to mention the paper that actually prompted that long ramble.

Axolotls are a fairly popular laboratory animal; they are salamanders, famous for their ability to regenerate limbs and for neotony — at least, they’re famous in the limited social circles where neotony can be a claim to fame. They’re also known (in even more limited circles) to be immunodeficient: While they do have all the components of an adaptive immune system, their antibody and T cell responses are remarkably slow and ineffective:1

Indeed, urodele amphibians do not react to soluble antigens. Their humoral immune responses are mediated by one unique class, the IgM, and are not anamnestic. A cellular co-operation has not been demonstrated during this humoral immune response; on the contrary, thymectomy, X-ray irradiation or corticosteroid treatment enhances the humoral immune response. Their MLR are usually very poor, and their transplantation reactions are chronic (21 days median survival time) …

(It may be worth noting that in the limited number of amphibians that have been studied to date — i.e. Xenopus — the larval form and the adult form have rather different immune systems; since the axolotl is permanently in a “larval” state, perhaps its immune system is also “immature”? However, the Xenopus larval immune system is apparently more effective than the axolotls’, so who knows.)

At any rate, it had been suggested that axolotl immune responses were defective because they have limited MHC diversity. Aside from the graft rejection, the logic of this link is not at all clear to me. Why would lack of diversity cause a slow antibody response in a single individual? The original paper says

The result of this non-diverse antigenic presentation could be a poor T-helper stimulation (considering the numher of different T clones stimulated), resulting in an extremely low cytokine synthesis and the absence of any cellular co-operation.

–which makes little sense to me; the authors seem to have confused population diversity with individual diversity.

Blogging on Peer-Reviewed ResearchAnyway, it’s all moot, because (and here after two posts I finally reach my original point) the limited diversity seems to be mainly because the original authors sampled laboratory axolotls, which are of course mostly derived from a handful of founders. A recent paper shows that wild axolotls — even though they are a small, critically-endangered population that went through a population bottleneck — have reasonably normal diversity.2 This paper is itself based on a small number of individuals — just nine — and although the sample size makes it hard to be sure I’d guess there is less diversity than “normal” species, but it’s clearly more than was originally claimed back in 1998:

Our results clearly overturn the supposition regarding lack of DAB polymorphism in A. mexicanum. Evidence for balancing selection summarized above also casts doubt on the more general proposition that this species is immunodeficient, although the small sample size limits the strength of this conclusion.

  1. Tournefier, A., Laurens, V., Chapusot, C., Ducoroy, P., Padros, M. R., Salvadori, F. et al. (1998). Structure of MHC class I and class II cDNAs and possible immunodeficiency linked to class II expression in the Mexican axolotl. Immunol Rev, 166, 259-277.[]
  2. Richman, A.D., Herrera, G., Reynoso, V.H., Mendez, G., Zambrano, L. (2007). Evidence for balancing selection at the DAB locus in the axolotl, Ambystoma mexicanum. International Journal of Immunogenetics, 34(6), 475-478. DOI: 10.1111/j.1744-313X.2007.00721.x[]
December 24th, 2007

MHC is diverse, except when it’s not

CheetahThe major histocompatibility complex is by far the most diverse region of vertebrate genomes; except when it isn’t. Since on the one hand it’s generally accepted that MHC genes are diverse in order to protect against pathogens, and on the other hand the precise mechanism driving diversity is controversial,1 it’s interesting to look at the species in which MHC is not particularly diverse. Are they particularly susceptible to pathogens? And is there an explanation for the lack of diversity?

I’ve mentioned a number of cases previously. Tasmanian Devils apparently have quite limited MHC diversity ,2 as do a number of other species including giant pandas,3 European beavers,4 Spanish ibex,5 and, perhaps most famously, cheetahs.

Most of these species are, as you can see, endangered. There are likely two important reasons for that: (1) Population bottlenecks lead to reduced genetic diversity, so endangered species (which have presumably been through a bottleneck relatively recently) are likely to have limited diversity; and (2) It’s easier to get funding to sequence the MHC from charismatic endangered species than from dirt-common starlings or what-not.

However, bottlenecks don’t entirely address the issue. I’ve previously mentioned the San Nicolas Island fox (Urocyon littoralis dickeyi), which has rather impressive MHC diversity despite undergoing drastic population bottlenecks within the past few hundred years.6

Although I believe the diversity at the MHC of these fox populations is still relatively low, it’s probably greater than some of the other populations I’ve mentioned here, such as the Tasmanian Devil, which have larger populations now and historically had more distant and less severe bottlenecks.

Cheetahs are the most famous example of an inbred population following a drastic population bottleneck; many people know that putatively-unrelated cheetahs can mutually tolerate skin grafts (a function of MHC similarity, of course),7 and lots of people also know that cheetahs are more susceptible to disease because of this limited diversity.

The problem with the latter “knowledge” is that as far I can find it’s not true — or at least, not demonstrated. It seems to be one of those circular things where the conclusion seems so obvious that it’s accepted with ridiculously inadequate evidence — whenever a cheetah gets sick, people nod wisely and point to the MHC, while when canine distemper wipes out half the lions in the Serengeti (leaving the cheetahs untouched)8 no one takes that as evidence for lion immunodeficiency. 9

Cottontop tamarinIn fact there’s ample evidence that populations with limited MHC diversity can do just fine out there. North American and European moose, 10 cotton-top tamarins, 11 and marmosets 12 have been claimed to have little MHC diversity. These species, like the cheetah, may indeed reflect limited populations a long time ago — moose apparently underwent a bottleneck around 10,000 years ago, about the same time as cheetahs; I don’t know about the other species — but the point is that no one is pointing to every sick moose and claiming that’s because of limited MHC diversity. What’s more, as the San Nicolas Island fox demonstrates, it’s possible to regenerate MHC diversity very fast — in hundreds rather than thousands of years; why didn’t cheetahs and moose do so? Probably because they didn’t need to, rather than couldn’t.

Limited MHC diversity is not an absolute barrier to species success. This should be pretty obvious, thinking about introduced species. There are over 100 million starlings in North America now, which arose from the 160 birds released just over 100 years ago in New York — how much MHC diversity could they possibly have? 13 Yet it was North American crows, not starlings, that were devastated by West Nile virus . Similarly, Australian rabbits, and rats around the world, have had huge populations arise from tiny numbers of founders, yet are not notorious for epidemics (except for artificially-introduced epidemics, in the case of Australian rabbits, and these are as remarkable for the rabbits’ resistance more than for their susceptibility).

So what’s the explanation? If pathogen pressure drives MHC diversity in almost all vertebrates, why do these vertebrates seem to do just fine with their limited diversity? Are these the exceptions that prove the rule, with hundreds of other introduced species disappearing because they didn’t have the right diversity? Are these species just the lucky ones, or just temporarily lucky and awaiting the right virus? I don’t know the answer, but if anyone has any ideas, let me know.

  1. Frequency-dependent selection or overdominant selection being the major two contenders; see here and links therein for more[]
  2. Siddle, H. V., Kreiss, A., Eldridge, M. D., Noonan, E., Clarke, C. J., Pyecroft, S. et al. (2007). Transmission of a fatal clonal tumor by biting occurs due to depleted MHC diversity in a threatened carnivorous marsupial. Proc Natl Acad Sci U S A. []
  3. Zhu, L., Ruan, X. D., Ge, Y. F., Wan, Q. H., & Fang, S. G. (2007). Low major histocompatibility complex class II DQA diversity in the Giant Panda (Ailuropoda melanoleuca). BMC Genet, 8, 29.[]
  4. Babik, W., Durka, W., & Radwan, J. (2005). Sequence diversity of the MHC DRB gene in the Eurasian beaver (Castor fiber). Mol Ecol, 14(14), 4249-4257.[]
  5. Amills, M., Jimenez, N., Jordana, J., Riccardi, A., Fernandez-Arias, A., Guiral, J. et al. (2004). Low diversity in the major histocompatibility complex class II DRB1 gene of the Spanish ibex, Capra pyrenaica. Heredity, 93(3), 266-272.[]
  6. Aguilar, A., Roemer, G., Debenham, S., Binns, M., Garcelon, D., and Wayne, R. K. (2004). High MHC diversity maintained by balancing selection in an otherwise genetically monomorphic mammal. Proc Natl Acad Sci U S A 101, 3490-3494. []
  7. O’Brien, S. J., Roelke, M. E., Marker, L., Newman, A., Winkler, C. A., Meltzer, D. et al. (1985). Genetic basis for species vulnerability in the cheetah. Science, 227(4693), 1428-1434.[]
  8. Guiserix, Micheline, Narges Bahi-Jaber, David Fouchet, Frank Sauvage, and Dominique Pontier. 2007. The canine distemper epidemic in Serengeti: are lions victims of a new highly virulent canine distemper virus strain, or is pathogen circulation stochasticity to blame? Journal of the Royal Society 4, no. 17 (December 22): 1127-34.[]
  9. I should say that I’m not saying wild-animal veterinarians or zoo vets do this — these are relatively lay people who I have talked with over the years. However, I’ve also seen papers that blithely talk about cheetahs being “unusually susceptible” — and what does “unusually susceptible” mean, anyway? Compared to some other species? What would be “usual”? — to diseases, citing single cases in zoo cheetahs as evidence — which is utterly ridiculous. E.g. Munson, Linda, Laurie Marker, Edward Dubovi, Jennifer A. Spencer, James F. Evermann, Stephen J. O’Brien, et al. 2004. SEROSURVEY OF VIRAL INFECTIONS IN FREE-RANGING NAMIBIAN CHEETAHS (ACINONYX JUBATUS). J Wildl Dis 40, no. 1 (January 1): 23-31. []
  10. Mikko, S. & Andersson, L. (1995). Low major histocompatibility complex class II diversity in European and North American moose. Proc Natl Acad Sci U S A, 92(10), 4259-4263.[]
  11. Gyllensten, U., Bergstrom, T., Josefsson, A., Sundvall, M., Savage, A., Blumer, E. S. et al. (1994). The cotton-top tamarin revisited: Mhc class I polymorphism of wild tamarins, and polymorphism and allelic diversity of the class II DQA1, DQB1, and DRB loci. Immunogenetics, 40(3), 167-176.[]
  12. Antunes, S. G., de Groot, N. G., Brok, H., Doxiadis, G., Menezes, A. A., Otting, N. et al. (1998). The common marmoset: a new world primate species with limited Mhc class II variability. Proc Natl Acad Sci U S A, 95(20), 11745-11750.[]
  13. No one seems to have looked, actually, and it would be interesting to have some numbers on this.[]
December 21st, 2007

Chicken MHC: Sloppy binding and evolutionary questions

13 day chick embryo
13 day chick embryo

There are a bunch of really interesting articles I want to talk about, but most will have to wait until after the holidays; with one sister and a brother visiting along with their assorted families, and my own Christmas responsibilities (including a three-year-old who confidently expects a motorcycle for Christmas: “But Santa SAID!”), I don’t have much time for detailed commentary.

One paper I do want to comment on, though, is
Koch, M., Camp, S., Collen, T., Avila, D., Salomonsen, J., Wallny, H.-J. et al. (2007). Structures of an MHC Class I Molecule from B21 Chickens Illustrate Promiscuous Peptide Binding. Immunity, 27(6), 885-899.

I may be mistaken, but I believe this is the first non-mammalian MHC class I molecule for which a crystal structure is available. (Actually, it’s not available, because the information they deposited in the Protein Data Bank is not yet released, even though the paper is out.) There are a number of interesting aspects about this molecule, but one is that it apparently violates one of the aspects of MHC that I considered (without thinking very much about it) to be a fairly fundamental characteristic. As such, it raises a question about MHC evolution that hadn’t previously occurred to me.

MHC class I binds to peptides, so that cytotoxic T lymphocytes can examine them. Mammalian MHC class I alleles (that have been tested) all bind to peptides with a broadly similar mechanism — much of the strength of the interaction comes from generic features that any peptide would have (the main chain) but there are also features that confer a certain amount of specificity to the interaction. Typically, a mammalian MHC class I molecule will bind to peptides that are about 9 amino acids long; two or three of those amino acids will snap, Lego-like, into an appropriate notch in the MHC. (See this post for more detail.) As a result, each MHC class I molecule can bind to many different peptide sequences, but only a limited subset of the large universe of 9-amino-acid long peptides. It’s this specificity that makes CTL epitope prediction possible. (However, it’s unusual but not extraordinary to identify peptides that bind, but don’t match the defined motif. For example, the exhaustive study of LCMV by Kotturi et al1 picked up two non-canonical peptides, out of the the 27 total known peptides for LCMV.)

Blogging on Peer-Reviewed ResearchThe interesting thing about this particular chicken MHC class I allele2 is that it seems to be much sloppier (“promiscuous”) than the mammalian alleles that have been looked at so far: “This molecule has a novel mode of peptide binding that allows peptides with completely different sequences to be presented to T lymphocytes.” Several other chicken alleles seem to also show promiscuous peptide binding, though other alleles probably are more conventional (or at least mammal-like) and have clear motifs.3

As a side note, this MHC class I allele is strongly associated with resistance to Marek’s Disease (a herpesvirus of chickens),4 and the authors here suggest that the promiscuous peptide-binding of B21 allows this allele to present more peptides from Marek’s disease, conferring greater resistance:

.. it is clear that many more peptides from representative Marek’s disease virus (MDV) genes are predicted to bind the BF2*2101 molecule than the MHC class I molecules from haplotypes such as B4, B12, and B15 that do not confer strong resistance to Marek’s disease. On this basis, it is likely that the promiscuous BF2*2101 molecule would have a greater chance of binding key protective peptide(s) than the fastidious MHC class I molecules from the other haplotypes.

Koch et al Fig 2
Chicken MHC BF2*2101

I am rather skeptical about this as the cause of resistance to Marek’s Disease. In the case of rapidly-mutating viruses like HIV, having a broad CTL response may well be correlated with resistance, but for herpesviruses, which are very stable genomically, even a single peptide epitope response can certainly confer protection (at least in mice); and it would be really surprising (even for a very “fastidious” allele) that there would be nothing at all (herpesviruses are big viruses, with lots of proteins) that binds. It’s possible, but I would look at other things first. 5

Back to my original issue. I’ve talked (ad nauseum!) about the evolution of MHC diversity. Very briefly6 MHC genes have by far the most alleles of any genes, and that is true for virtually every species that’s been examined — fish, birds, reptiles, mammals. There are two widely-accepted explanations for this diversity: Overdominance, or frequency-dependent selection. (Frequency-dependent selection seems to have a little more evidence on its side.) Both explanations depend on different MHC alleles presenting different peptide subsets from pathogens.

Now here’s my question, and in light of the chicken BF2*2101 data I don’t have an obvious answer for it. (This may be because of my tinsel-addled state, and maybe once the holidays are over the explanation will be obvious to me.) Why do MHC alleles bind to peptide subsets? What is the advantage of binding to subsets, compared to binding to all peptides? If MHC can bind promiscuously to many different peptides, then pathogens would not be able to escape from the MHC. For example, we would have much greater resistance to HIV, because there would be no escape mutants.

The explanation I used to have is that if MHC binds to all peptides, then there would be too many self peptides presented, and the process of negative selection in the thymus would eliminate too many T cells during their maturation. In other words, MHC alleles have to trade off the ability to present many pathogen peptides, in order to leave gaps in the self-reactive repertoire for T cells to actually develop.

But these B21 chickens can apparently get away with promiscuous peptide binding. How does that work? I am suddenly very curious about thymic selection in these chickens. What kind of TcR repertoire do they have? I wonder if they’ve looked.

  1. Kotturi, M. F., Peters, B., Buendia-Laysa, F. J., Sidney, J., Oseroff, C., Botten, J. et al. (2007). The CD8+ T-cell response to lymphocytic choriomeningitis virus involves the L antigen: uncovering new tricks for an old virus. J Virol, 81(10), 4928-4940. []
  2. BF2*2101[]
  3. Wallny H, Avila D, Hunt LG, Powell TJ, Riegert P, Salomonsen J, et al. Peptide motifs of the single dominantly expressed class I molecule explain the striking MHC-determined response to Rous sarcoma virus in chickens. Proceedings of the National Academy of Sciences of the United States of America. 2006 31;103(5):1434-9.
    Kaufman J, Milne S, Gobel TWF, Walker BA, Jacob JP, Auffray C, et al. The chicken B locus is a minimal essential major histocompatibility complex. Nature. 1999 Oct 28;401(6756):923-925.[]
  4. Briles WE, Stone HA, Cole RK. Marek’s disease: effects of B histocompatibility alloalleles in resistant and susceptible chicken lines. Science (New York, N.Y.). 1977 14;195(4274):193-5.[]
  5. There is a little precedent for the suggestion, in that it’s been proposed that Epstein-Barr virus, a human herpesvirus, evolved to escape binding by a prevalent MHC class I allele in a particular population: de Campos-Lima, P. O., Gavioli, R., Zhang, Q. J., Wallace, L. E., Dolcetti, R., Rowe, M. et al. (1993). HLA-A11 epitope loss isolates of Epstein-Barr virus from a highly A11+ population. Science, 260(5104), 98-100 and several more recent studies. I am not overwhelmed by those studies either, although the latest (Midgley, R. S., Bell, A. I., McGeoch, D. J., & Rickinson, A. B. (2003). Latent gene sequencing reveals familial relationships among Chinese Epstein-Barr virus strains and evidence for positive selection of A11 epitope changes. J Virol, 77(21), 11517-11530. ) is the most convincing.[]
  6. See here, here, here, and here for more detail, if you care[]
December 16th, 2007

Malaria eradication: The smallpox precedent

Tametomo's force driving away the gods of smallpox. Yoshitoshi Taiso, 1890
Tametomo’s force driving away the gods of smallpox. Yoshitoshi Taiso, 1890

Following up to my last post , about the Gates Foundation’s call to eradicate malaria, I thought I would talk about historical experience with eradication of infectious diseases. Here is the list of diseases that have been eradicated throughout all of recorded history:

  1. Smallpox

I’ll pause so you can write that down.

OK, there are a couple of other diseases that are, hopefully, on their way to eradication (notably poliovirus), and there are a bunch of others whose incidence has been spectacularly reduced through vaccination (such as measles, diphtheria, and rubella),1 sanitation (such as guinea worm), and even antibiotics (leprosy). But only smallpox has been eradicated. 2

Why was smallpox eradicated, where four other global eradication campaigns3 failed? What was special about smallpox and its vaccine? What are the factors that allowed this disease to be reduced from millions of cases per year, to none? And, most to the point, what aspects of smallpox eradication are applicable to malaria?

In fact, most of the special aspects of smallpox that allowed it to be eradicated are not particularly true for malaria. Smallpox …

  • Has no animal host. If you can eradicate the disease in humans, it won’t re-emerge from a mouse, or monkey, or bat reservoir — compare to yellow fever, for example.
  • Has no persistent phase. Smallpox either kills people, or they recover completely and eliminate the virus. In either case, if there are no clinical cases over a reasonable period, then you can be confident that there is no more virus.
  • Induces long-term immunity in survivors.
  • Was a fearful enough disease that the political will to eradicate it lasted through the campaign. Smallpox vaccination continued throughout civil wars and other upheavals.
 Vaccinating the poor / Drawn by Sol Ettinge, Jun. 1872
Vaccinating the poor. Sol Ettinge, Jr., 1872

And the smallpox vaccine (vaccinia virus) is also exceptional in that it …

  • Induces very long-term immunity with a single dose. Vaccinia virus induces a memory, and probably protective, immune response for an extraordinarily long time — response have been shown for up to 60 years.
  • Is relatively stable and easy to transport and deliver. With large-scale vaccination campaigns, logistics become the limiting factor, especially as the campaign progresses and the final reservoirs of disease may be in remote, third-world areas.
  • Leaves a marker of treatment. Vaccinated people usually had a small scar at the site of scarification, so that it was possible to identify susceptible people and protect them.

The smallpox vaccine is also exceptional in its frequency and severity of adverse effects. I think that for no disease today would the risks of smallpox vaccine be tolerated — back to the fourth point above, that smallpox was such a terrible disease that people were willing to take the risks of vaccination. 4

There were also a vast number of technical and logistic components that, I think, are mostly applicable to any eradication program (for example, the cost per dose of a vaccine is much less if the vaccine can be prepared in large, multi-dose vials; but that means you need to use the vial up all at once, which means in turn organizing large numbers of vaccination on a single day; and that in turn implies an efficient communication network and so on), and which I won’t talk about here. There’s a fascinating review in Henderson, D. A. (1987). Principles and lessons from the smallpox eradication programme. Bull World Health Organ, 65(4), 535-546. if you want to learn more.

“A much greater change — not apparent but real — was produced by the introduction of vaccination in 1798. It was computed, that, in 1795, when the population of the British Isles was 15,000,000, the deaths produced by the small-pox amounted to 36,000, or nearly 11 per cent. of the whole annual mortality. Now, since not more than one case in 330 terminates fatally under the cow-pox system, either directly by the primary infection, or from the other diseases supervening; the whole of the young persons destroyed by the small-pox might be considered as saved, were vaccination universal, and always properly performed. This is not precisely the case, but one or one and a half per cent. will cover the deficiencies; and we therefore conclude, that vaccination has diminished the annual mortality fully nine per cent. After we had arrived at this conclusion by the process described, we found it confirmed by the authority of Mr Milne, who estimates, in a note to one of his tables, that the mortality of 1 in 40 would be diminished to 1 in 43-45, by exterminating the small-pox. Now this is almost precisely 9 per cent.”
Combe, George. 1847. The Constitution of Man and Its Relation to External Objects. Edinburgh: Maclachlan, Stewart, & Co., Longman & Co.; Simpkin, Marshall, & Co., W. S. Orr & Co., London, James M’Glashan, Dublin.
It’s important to point out that eradication of a disease is possible when not all of these factors are matched — poliovirus, which is almost eradicated (and could have been eradicated altogether with a bit more political help) is different in several ways. But it does offer a checklist for known success. How does malaria match up?

Not so well, actually. Malaria …

  • May have an animal reservoir. Apes can be infected experimentally, and are sometimes naturally infected. This is not a practical issue today, where the animal reservoir is negligible, but if human infection is reduced an animal reservoir might serve as a source for reinfection.
  • Does have a persistent phase. This is especially a concern since partially-immune people (common in endemic areas) can be infected and trasmit the disease without showing clinical symptoms — again, a potential reservoir of re-infection.
  • Does not consistently induce protective immunity.
  • Is a terrible scourge, but one to which the world has become accustomed. Is there the will to take on the cost of eradication? The last attempt at malaria eradication — which failed — cost a billion dollars. As Melinda Gates pointed out, the cost of the disease in perpetuity is greater than the cost of eradication, but the costs come from different places.

Since there are no effective malaria vaccines as yet, we can’t very well compare them to the smallpox vaccine. I don’t know enough about the irradiated vaccine that will enter trials next year, but the “RTS,S/AS02D” vaccine in phase I/II trials5 requires multiple doses and apparently offers relatively low protection — certainly better than nothing, if this holds true through phase III trials, but it’s hard to imagine that it’s sufficient for eradication.

So vaccines are probably going to be an important component of malaria eradication (if it happens) but the nature of the disease means that they’re not likely to be sufficient. Melinda Gates said in her eradication speech that “This is a long-term goal; it will not come soon,” and she focused on four “intervention points”:

To eradicate malaria, you have to end transmission — and there are multiple points where you can intervene. Reduce the number of infected mosquitoes. Keep mosquitoes from biting people. Keep people who are bitten from getting infected. Keep people who are infected from transmitting malaria back to mosquitoes.

Vaccines are good candidates to help with the last two points, and may help with the first. But overall, this is a more complex problem than smallpox. Nevertheless, smallpox eradication has plenty of lessons for malaria, as well.

  1. A great review, with dramatic incidence tables is: Roush, S. W. & Murphy, T. V. (2007). Historical comparisons of morbidity and mortality for vaccine-preventable diseases in the United States. JAMA, 298(18), 2155-2163. I have adapted their numbers to make a table here []
  2. It is probably true that there are stocks of the virus around as well as the official stocks. However, there have been no cases of “wild” human smallpox since 1977.[]
  3. Henderson, D. A. (1999). Lessons from the eradication campaigns. Vaccine, 17 Suppl 3, S53-5. []
  4. Belongia, E. A. & Naleway, A. L. (2003). Smallpox vaccine: the good, the bad, and the ugly. Clin Med Res, 1(2), 87-92.[]
  5. Aponte, J. J., Aide, P., Renom, M., Mandomando, I., Bassat, Q., Sacarlal, J. et al. (2007). Safety of the RTS,S/AS02D candidate malaria vaccine in infants living in a highly endemic area of Mozambique: a double blind randomised controlled phase I/IIb trial. Lancet, 370(9598), 1543-1551.[]
December 12th, 2007

Malaria eradication?

Eradicate Malaria India 1958 I’m marking final exams for the grad immunology class I teach, so I don’t have a lot of time to blog. But I do want to point to a really amazing, ambitious, and potentially world-changing initiative that doesn’t seem to have got the attention it deserves in the blog-world. A couple of months ago, Melinda Gates made a speech in which she said:

Bill and I believe that these advances in science and medicine, your promising research, and the rising concern of people around the world represent an historic opportunity not just to treat malaria or to control it-but to chart a long-term course to eradicate it.

I don’t need to give figures, I think, on what a devastating disease malaria is. The WHO fact sheet is filled with dismal stats (“A child dies of malaria every 30 seconds.“) And I’ve previously blogged about the track record of malaria vaccines, which have been encouraging but unsuccessful for forty years. Gates’ proposal really is (as she herself says) audacious, but I think she presents three excellent reasons for aiming for eradication:

  • the human cost of malaria
  • the financial cost. “If we plan only to control malaria, we will never eradicate it.
  • history, which tells us that any malaria control is just temporary: “the ability of the parasite to develop resistance to insecticides and medicines tells us that no set of control strategies can control malaria for very long.

Blogging on Peer-Reviewed ResearchProtect against malaria 1941 Is it possible? I have no idea, myself. It’s been tried before (the poster at the top is from 1958) without success, and we are certainly a long, long way away from that aim at the moment. I do think it’s a worthy goal. And there are some new glimmers of hope. A Lancet article1 that came out about the same time as Gates’ talk shows that a new malaria vaccine is safe and at least moderately effective.

Is “moderately effective” good enough? We don’t really know yet how effective the vaccine is; this study (which wasn’t designed to test effectiveness per se) found around a 65% level of protection — low for a vaccine in general; high for a malaria vaccine. A commentary on the paper in the same issue of Lancet2 says that “Some experts have predicted that the effect of the introduction of a partly protective vaccine will be reduction in morbidity and mortality in the first years of life, with negligible effect on transmission.” If so, then this is more a step toward control than eradication.

Still, it’s a step, and if in fact vaccination can reduce malaria at all then it’s a very promising step. Other vaccines are on their way, and the experience with this one will help in developing more and more effective approaches. As Epstein’s commentary3 says, “The next 5-10 years will probably be the most exciting in the long journey to bring a malaria vaccine to the developing world.

  1. APONTE, J., AIDE, P., RENOM, M., MANDOMANDO, I., BASSAT, Q., SACARLAL, J., MANACA, M., LAFUENTE, S., BARBOSA, A., LEACH, A. (2007). Safety of the RTS,S/AS02D candidate malaria vaccine in infants living in a highly endemic area of Mozambique: a double blind randomised controlled phase I/IIb trial. The Lancet, 370(9598), 1543-1551. DOI: 10.1016/S0140-6736(07)61542-6[]
  2. What will a partly protective malaria vaccine mean to mothers in Africa?Judith E Epstein The Lancet 370, 3 November 2007-9 November 2007, Pages 1523-1524 []
  3. What will a partly protective malaria vaccine mean to mothers in Africa? Judith E Epstein. The Lancet 370, 3 November 2007-9 November 2007, Pages 1523-1524 []
December 9th, 2007

HIV and T cell activation

HIV virion One of the long-standing puzzles in the pathogenesis of AIDS is exactly why, in fact, untreated HIV leads to the inexorable loss of helper (CD4+) T cells that eventually manifests itself as immunosuppresssion.

It’s not that there are no explanations for the loss — rather there are too many possible explanations, and identifying the most important cause hasn’t been easy. Is it because the virus directly destroys the cells it infects? Do HIV-specific cytotoxic T cells recognize and destroy infected cells? Are particular subsets especially vulnerable, so that the obvious loss of circulating cells is just an indicator of the true underlying problem? All these are probably true, and may be contributers — even important contributers — to the CD4 cell loss. 1 But over the past few years, a bunch of experiments have shown that these more traditional causes are probably not enough to account for the observed loss. (For example, far more CD4 cells die than are ever infected with HIV.)

Instead, the chronic activation of T cells in HIV-infected humans seems to be particularly important. In a normal immune response, T cells recognize their target, become activated, divide and expand a thousand-fold, and then contract again. Obviously, if T cells just expanded every time we mount an immune response, we would quite soon consist of nothing but T cells, so the contraction phase — a phase of the normal immune response in which T cells die off — is critical to reset T cell numbers to something appropriate once again. Therefore, a normal consequence of T cell activation is T cell death. This can happen many times, and our overall T cell numbers remain remarkably constant; there is some kind of feedback mechanism that counts T cells and ensures that there aren’t too many or too few.

What happens if, rather than regular but limited periods of activation each time we encounter a new virus or whatever, we experience a constant activation of a large number of T cells at once? There would be a constant death of T cells as well. Experiments show that when this happens, the feedback mechanism is not perfect; more cells can be lost than are replaced.

T cell activation

Immune activation is a hallmark of HIV infection, and the rate and extent of CD4 T cell loss correlates very well with this activation. The first time I was exposed to this concept was in 2002,2 though I don’t know if this was the first time it was suggested. A very recent paper in Journal of Infectious diseases shows that even with very low levels of HIV viremia, T cell activation is correlated with CD4 T cell loss.3

Is this because there is a widespread immune response targeting HIV? Are the activated CD4 T cells all specific for HIV? That’s the simplest explanation, but it’s not the right one; few of the activated cells are specific for HIV.

Blogging on Peer-Reviewed ResearchA plausible alternative explanation was “bystander activation” — a spillover of the activation event, which is normally very closely targeted to a small number of specific T cells, into the large pool of neighboring T cells that are not specific for the original activating event.4 So is the chronic exposure to HIV causing bystander activation of neighboring cells, which then die? Probably not. “Bystander activation” was a popular concept in the early 1990s or so, but I think it was never universally accepted, and when tools to measure specificity of the activated cells became available (in the mid-1990s) it was quickly shown that almost all activation was in fact specific. Bystander activation probably does exist, but it’s a minor effect.

There are other possibilities. Perhaps HIV is killing or otherwise screwing up regulatory T cells (TRegs) — which are CD4 T cells — and these are no longer able to perform their usual function of tempering immune activation. Perhaps the rapid early loss of gut T cells results in chronic infection in the gut. Or perhaps there’s an autoimmune component to the activation.5

One reason this may be particularly relevant right now is the recent HIV vaccine fiasco , which has been widely discussed. (A particularly excellent discussion is at the Michael Palm Treatment Action Group blog, starting here and with many followup posts, especially including this one and this one .) Briefly, it seems possible that activation of the immune system by the vaccine — the generic adenovirus part, rather than the HIV-specific part — may have actually enhanced disease. It certainly sounds familiar, but it may be unrelated to the activation-induced cell death associated with AIDS progression.

  1. An important point is that cell death directly because of HIV infection may well be important in some subsets — in particular, gut T cells are probably rapidly killed by HIV infection. But the cells in circulation, that correlate with the progression to AIDS, are not.[]
  2. Sousa, A. E., Carneiro, J., Meier-Schellersheim, M., Grossman, Z., & Victorino, R. M. (2002). CD4 T cell depletion is linked directly to immune activation in the pathogenesis of HIV-1 and HIV-2 but only indirectly to the viral load. J Immunol, 169(6), 3400-3406.[]
  3. Relationship between T Cell Activation and CD4+ T Cell Count in HIV‐Seropositive Individuals with Undetectable Plasma HIV RNA Levels in the Absence of Therapy. Peter W. Hunt, Jason Brenchley, Elizabeth Sinclair, Joseph M. McCune, Michelle Roland, Kimberly Page‐Shafer, Priscilla Hsue, Brinda Emu, Melissa Krone, Harry Lampiris, Daniel Douek, Jeffrey N. Martin, Steven G. Deeks. The Journal of Infectious Diseases 2008;197 []
  4. Bangs, S. C., McMichael, A. J., & Xu, X. N. (2006). Bystander T cell activation–implications for HIV infection and other diseases. Trends Immunol, 27(11), 518-524. []
  5. Rawson, P. M., Molette, C., Videtta, M., Altieri, L., Franceschini, D., Donato, T. et al. (2007). Cross-presentation of caspase-cleaved apoptotic self antigens in HIV infection. Nat Med. []
December 5th, 2007

Bioweaponized wasps

Braconid wasp When I am exposed to mystery rays from outer space and develop superpowers, I want it to involve a symbiotic virus that I can use to attack my foes. I swear that I will use this power only for good. 1

Braconid wasp and prey Of course, I’ve been beaten to the big cartoon punch. When I first heard about polydnaviruses, about ten years ago, I realized that I could never be as cool as a microgastroid wasp.2 Microgastrinae and Campopleginae are parasitoid wasps that have (apparently independently) managed to set up a spectacular symbiotic relationship with polydnaviruses. The wasps are predators of other insects; they lay their eggs on caterpillars or aphids or whatever, the eggs hatch, and the wasp larvae eat their host from the inside out, finally emerging as new adult wasps.

Understandably, the host caterpillar is not all that enthusiastic about this process, and attempts to defend themselves with an immune response to the wasp larvae. In an effective immune response against endoparasitoids, the host’s immune cells (hemocytes) will encapsulate the parasite, wrapping it up and walling it off from the host. 3 Successful parasitic wasps (of which there are a huge number; this is a very popular ecological niche) must avoid their dinner’s immune system, and there are many different routes to to this goal.

Rwandan stampNow hold that thought for a moment while I change the subject to viruses. One characteristic of a successful virus is that it must, to some extent, avoid its host immune system. I’ve talked about that in mammalian viruses , and insect viruses are in principle no different. Insects don’t have an adaptive immune system (though see some discussion of that here and here), and insect viruses therefore have to deal with mechanisms like recognition and encapisidation by hemocytes.

Blogging on Peer-Reviewed ResearchYou see where this is going. Some 75 million years ago,4 wasps set up a symbiosis with viral pathogens of the wasps’ prey. The wasps use the virus’s immune evasion functions — of which there are a huge number5 and which, as well as being interesting in their own right, have obvious potential uses in pest control — to avoid elimination by their host’s immune system. And what do the viruses get out of it? They get free replication out of it: They’ve become completely integrated into the wasp genome. They’re no longer a free-living6 entity; they do not replicate in the cells of the host, nor do they replicate independently in the wasp cells. The only way the viral genome can make a new viral genome is in the genome of the wasp that they are helping parasitize a new host.

The symbiosis model is the traditional explanation, but it’s not the only one. There are at least two, probably three, and maybe more, lineages of polydnavirus-carrying wasps, and the polydnavirus sequences seem to be very different, suggesting that the wasp lineages arose independently. For some of these, there is at least some evidence that the polydnaviruses arose from free-living viruses;7 but for other it’s been suggested that the polydnaviruses actually represent expansion of wasp genes that then grabbed a capsid protein and became virus-like after the fact: 8

A more parsimonious hypothesis would be that bracoviruses do not originate from any of the large genome viruses characterized to date. They may have been built up from a simple system producing circular DNA intermediates, such as mobile elements, within the wasp genome. The acquisition of a capsid protein, possibly of viral origin, around the circular DNA intermediates would have allowed infection of lepidopteran cells. Finally, virulence genes could have been acquired from the wasp genome at different times during evolution of bracovirus-bearing wasp lineages, thus explaining why CcBV genes encoding proteins with a predicted function resemble cellular genes.

In any case, this approach has turned out to be incredibly successful, with tens of thousands of parasitic wasps making use of these bioweapons.

  1. Although, you know that guy with the hat? With his blinker on, driving 45 mph in the fast lane? PEW! PEW! PEW![]
  2. Or, for that matter, as a campoplegid wasp.[]
  3. A nice review of the insect immune response to endoparasitoids is Schmidt O, Theopold U, Strand M. Innate immunity and its evasion and suppression by hymenopteran endoparasitoids. BioEssays. 2001 ;23(4):344-351.[]
  4. Whitfield, J.B. Estimating the age of the polydnavirus/braconid wasp symbiosis. Proc Natl Acad Sci U S A 99, 7508-7513 (2002).[]
  5. Recent examples include — Beck, M.H. & Strand, M.R. A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway. Proc Natl Acad Sci U S A doi:10.1073/pnas.0708056104 (2007).
    Thoetkiattikul, H., Beck, M.H. & Strand, M.R. Inhibitor kappaB-like proteins from a polydnavirus inhibit NF-kappaB activation and suppress the insect immune response. Proc Natl Acad Sci U S A 102, 11426-11431 (2005). []
  6. to the extent any virus can be free living[]
  7. 1. Lapointe, R. et al. Genomic and Morphological Features of a Banchine Polydnavirus: Comparison with Bracoviruses and Ichnoviruses. The Journal of Virology 81, 6491-6501 (2007).[]
  8. Espagne, E. et al. Genome sequence of a polydnavirus: insights into symbiotic virus evolution. Science 306, 286-289 (2004).[]
December 2nd, 2007

Same trip, different routes: Lamprey immunity

Placoderm A few years ago, one of the aspects of the immune system that puzzled me most was the shark immune system. It’s not that sharks have an immune system; everything has some form of resistance against parasites, or else they don’t exist any more. Nor is it surprising that sharks’ immune systems are really pretty similar to ours: They have T cell receptors that are generated through somatic recombination, they have MHC class I and II, they have T cells and B cells and a thymus — not to mention all the innate stuff that is also pretty similar. But sharks are really not all that far away from us, evolutionarily speaking, so it’s not astonishing that their immune systems are not far away either.

What was astonishing to me is that this all (apparently) arrived all at once. Lampreys and hagfish, with a common ancestor not far from sharks, did not (I thought) have any of those things. All the pieces, that need to fit precisely with each other to work at all, arose from nothing — an immunological Big Bang.1 That’s not how evolution is supposed to work.

I assured myself that this just seemed puzzling, and probably once people started looking more closely at lamprey or hagfish immunity we would find that the common ancestor really did have some of these things, that were co-opted into the jawed-vertebrate immune system. And in the past five years or so, that’s pretty much what has happened. We are still in early days of lamprey immunology, but already we can see much of the foundation of the shark (and our) immune systems there. Sea urchins (which have a common ancestor with lampreys) have molecules that are much like RAG. Lampreys have cells that are very lymphocyte-like.2 These cells accumulate in a region that looks quite thymus-like. Lampreys even have a proto-T cell receptor, and something very CD4-like,3 although as far as I know no MHC-like molecules have yet been identified.

Lamprey So lampreys have most, if not all, of the requirements for an adaptive immune system. In fact, lampreys apparently do have an adaptive immune system. The hallmark of an adaptive immune system is immunological memory, and lampreys act as if they have immune memory (though it’s not as fast as ours), responding more rapidly to antigens on a second exposure, 4 although I think some of the caveats that apply to the “insect immune memory” observations apply here as well.5

And yet there is still one big surprise. The most interesting part of the lamprey immune system is not so much the similarities to jawed vertebrates: it’s the differences. In sharks and their children, unto us, lymphocytes (T cells and B cells) make tremendously diverse receptors that can interact specifically with antigens. This diversity is produced using RAG recombination to make T cell receptors and antibodies. Lampreys also make an equally diverse, antigen-recognizing receptor in their lymphocytes — but their receptors look nothing like ours, and are generated using an entirely different system of somatic rearrangement!

Kim et al Fig 5 The antigen-specific receptor on lamprey (and hagfish) lymphocytes6 is called a “VLR” (variable leukocyte receptor), and it is not related in the least to TcRs or antibodies; both of the latter are built on the immunoglobulin fold, while VLRs are made of leucine-rich repeats that produce the characteristic horseshoe shape of an LRR. (Toll-like receptors are also members of the LRR family, so I wondered about the possibility of a TLR-like molecule having been co-opted from an innate to an adaptive role. But apparently VLRs are members of a different family of LRRs than are TLRs,7 being more similar to some platelet receptors in humans.) VLRs are, as I say, enormously diverse, at least as variable as are TLRs, and just as with TcRs their variable recgions are clustered, in what seems to be the antigen-recognition region (the figure at left, from Kim et al., 8 is of a hagfish VLR, with the variable regions indicated in red; click for a larger version.) Like TcRs, their diversity arises through DNA shuffling in somatic cells. But the mechanism of shuffling is different, involving combinatorial use of a large number of cassettes, or modules — some 2000 of them. 9

Blogging on Peer-Reviewed ResearchBut there’s more! This mechanism of diversity in lampreys, which really is quite different from the TcR mechanism of recombination? It uses a cytosine deaminase! Why is that interesting? 7 Because a related cytosine deaminase (AID-APOBEC) is involved in human immunoglobulin class switching and maturation! 10 So even though lampreys have developed a different mechanism of receptor diversity, they (and therefore, probably, our common ancestors) use some of the same molecules that we use in our receptor diversity system.

Five years ago, it seemed that the shark immunological Big Bang came out of nowhere. Now — exactly as we’d expect from evolution — we know that sharks adapted pre-existing systems, and that rather than a Big Bang there was more like a gradual accumulation of pieces that added up to a revolution.

  1. Bernstein, R. M., Schluter, S. F., Bernstein, H., and Marchalonis, J. J. (1996). Primordial emergence of the recombination activating gene 1 (RAG1): sequence of the complete shark gene indicates homology to microbial integrases. Proc Natl Acad Sci U S A 93, 9454-9459.[]
  2. Shintani, S., Terzic, J., Sato, A., Saraga-Babic, M., O’hUigin, C., Tichy, H., and Klein, J. (2000). Do lampreys have lymphocytes? The Spi evidence. Proc Natl Acad Sci U S A 97, 7417-7422.
    Mayer, W. E., Uinuk-Ool, T., Tichy, H., Gartland, L. A., Klein, J., and Cooper, M. D. (2002). Isolation and characterization of lymphocyte-like cells from a lamprey. Proc Natl Acad Sci U S A 99, 14350-14355.
    and especially
    Uinuk-Ool, T., Mayer, W. E., Sato, A., Dongak, R., Cooper, M. D., and Klein, J. (2002). Lamprey lymphocyte-like cells express homologs of genes involved in immunologically relevant activities of mammalian lymphocytes. Proc Natl Acad Sci U S A 99, 14356-14361. []
  3. Pancer, Z., Mayer, W. E., Klein, J., and Cooper, M. D. (2004). Prototypic T cell receptor and CD4-like coreceptor are expressed by lymphocytes in the agnathan sea lamprey. Proc Natl Acad Sci U S A 101, 13273-13278. []
  4. For example — I haven’t read these articles directly, but they are regularly cited in more recent papers — Perey, D. Y., Finstad, J., Pollara, B. & Good, R. A. Evolution of the immune response. VI. First and second set skin homograft rejections in primitive fishes. Lab. Invest. 19, 591-597 (1968)
    Fujii, T., Nakagawa, H., and Murakawa, S. (1979). Immunity in lamprey. I. Production of haemolytic and haemagglutinating antibody to sheep red blood cells in Japanese lampreys. Dev Comp Immunol 3, 441-451.[]
  5. But not all, because as you will see a mechanistic explanation for lamprey memory is taking shape.[]
  6. Pancer, Z., Amemiya, C. T., Ehrhardt, G. R., Ceitlin, J., Gartland, G. L., and Cooper, M. D. (2004). Somatic diversification of variable lymphocyte receptors in the agnathan sea lamprey. Nature 430, 174-180. []
  7. Rogozin, I. B., Iyer, L. M., Liang, L., Glazko, G. V., Liston, V. G., Pavlov, Y. I., Aravind, L., and Pancer, Z. (2007). Evolution and diversification of lamprey antigen receptors: evidence for involvement of an AID-APOBEC family cytosine deaminase. Nat Immunol 8, 647-656. [][]
  8. Kim, H. M., Oh, S. C., Lim, K. J., Kasamatsu, J., Heo, J. Y., Park, B. S., Lee, H., Yoo, O. J., Kasahara, M., and Lee, J. O. (2007). Structural diversity of the hagfish variable lymphocyte receptors. J Biol Chem 282, 6726-6732. []
  9. Nagawa, F., Kishishita, N., Shimizu, K., Hirose, S., Miyoshi, M., Nezu, J., Nishimura, T., Nishizumi, H., Takahashi, Y., Hashimoto, S., Takeuchi, M., Miyajima, A., Takemori, T., Otsuka, A. J., and Sakano, H. (2007). Antigen-receptor genes of the agnathan lamprey are assembled by a process involving copy choice. Nat Immunol 8, 206-213.
    Rogozin, I. B., Iyer, L. M., Liang, L., Glazko, G. V., Liston, V. G., Pavlov, Y. I., Aravind, L., and Pancer, Z. (2007). Evolution and diversification of lamprey antigen receptors: evidence for involvement of an AID-APOBEC family cytosine deaminase. Nat Immunol 8, []
  10. Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y. and Honjo, T. (2000). Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102:553-563. []