Mystery Rays from Outer Space

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

March 30th, 2008

What’s in a name? (Are cytotoxic T lymphocytes cytotoxic?)

T lymphocyte, SEMIn one of those bizarre twists of logic, cytotoxic T lymphocytes were so named because they’re T lymphocytes that are cytotoxic. Is that all they are?

Cytotoxicity is relatively easy to measure — there are straightforward ways to measure cell death, and it can be a nice, binary, black/white distinction. If you take lymphocytes from a mouse (or a person) that was previously infected with a virus, and you mix those lymphocytes with cells infected with the same virus, the infected cells will be killed. If you look at the surface markers of the cells responsible for the killing, you can narrow it down to T cells (i.e. with the T cell receptor) that have the CD8 surface marker. 1

51Cr release assays2 are a traditional way of measuring cell death, and you can set them up in 96-well plates and get moderate throughput to test multiple conditions. It’s a convenient system, and it was the first one to be widely used to T cells. Doherty and Zinkernagel used it in their Nobel-Prize winning work on MHC restriction, for example.

However, as you’d expect, systems which are designed for operator convenience don’t necessarily reflect reality. Measuring cell death in vivo, that is, inside a virus-infected animal, is much more complicated than in a 96-well plate. Do CTL actually kill in that context? And even if it does happen, is it the only thing that happens? Could CTL be doing something else during an infection, other than killing, that helps in their mission?

You might wonder if immunologists were blinkered by the name — how could cytotoxic T lymphocytes not be, first and foremost, cytotoxic? — but I don’t think it’s revisionist to say that’s not true. I think most of us were pretty sure that CTL had lots of other weapons in their arsenal, but how much other stuff? How often were CTL actually cytotoxic, and how often did they do other stuff?

One problem with cytotoxicity as an assay for this question, is that it’s a bulk assay. Until recently you couldn’t really measure killing by a single CTL. (You can now, though. Uli von Andrian has some beautiful videos of CTL punching holes in their targets here here, from his 1996 2006 Immunity paper. 3 Von Andrian’s site is filled with beautiful and amazing videos; check them out.). You mix together a batch of CTL with the targets — the targets die, well and good — but were all of the specific CTL helping out, or was it just the work of a minority of them that are specialized for killing?

In 1996, Mark Davis introduced a new and exciting technology, MHC tetramers, that’s able to rapidly identify T cells by phenotype rather than function. 4 That is, if a T cell has the right T cell receptor to recognize a virus-infected cell, tetramers can show you the T cell — even if it cannot kill. This was pretty revolutionary, and led to some drastic increases in estimates of T cell number — previous methods of counting specific T cells were known to be underestimates, and tetramer staining showed us that there were sometimes 100 or 1000 times more CTL floating around than had been esimated.

It didn’t really answer the cytotoxicity question, though. Tetramer staining correlates well with cytotoxicity levels, but you’d see that even if 1% of the CTL were actually cytotoxic, and the rest were doing something else.Intracellular cytokine ctaining

Another new technique that came out around the same time or a little later5 is intracellular cytokine staining. This identifies T cells that not only recognize their target, but react to it by producing cytokines, such as interferon. In other words, intracellular cytokine staining not only lets you measure T cells, it offers a measure of functionality other than cytotoxicity. Correlating this with tetramer staining was a little more informative; most tetramer-positive cells were also able to produce interferon, for example.

So we know that there are a lot of CTL; we knew that most produced interferon and other cytokines when stimulated. But — to finally get to the point — we also knew even by the that cytotoxicity is important. Just about the same time as all these other assays were coming out, a perforin knockout mouse was made. Perforin is a protein that’s believed to be important in CTL cytotoxicity and not much else.6 Even though other proteins are also involved in cytotoxicity, mice without perforin weren’t able to clear lymphocytic choriomeningitis virus the way wild-type mice did. 7

So what’s the interferon there for? Interferon isn’t directly involved in cytotoxicity, and experiments from around that time showed that CTL can do a lot of antiviral work just using interferon, without getting all cytotoxic on us. I was originally going to talk about that experiment — Frank Chisari’s hepatitis B mouse model — here, but this is all background, so I’ll get to that some other day.

  1. Also, probably, you’ll find that natural killer, NK, cells do some killing too.[]
  2. Which really suck, but they’re better than the alternatives, which suck even more[]
  3. Mempel, T. R., Pittet, M. J., Khazaie, K., Weninger, W., Weissleder, R., von Boehmer, H., and von Andrian, U. H. (2006). Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation. Immunity 25, 129-141. doi:10.1016/j.immuni.2006.04.015 []
  4. Altman, J.D., Moss, P.A., Goulder, P.J., Barouch, D.H., McHeyzer-Williams, M.G., Bell, J.I., McMichael, A.J., Davis, M.M. (1996). Phenotypic Analysis of Antigen-Specific T Lymphocytes. Science, 274(5284), 94-96. DOI: 10.1126/science.274.5284.94[]
  5. I confess I’m not quite sure when it was developed — it became popular in the mid- to late-90s, is all I remember. The assay is basically a spinoff of the earlier ELISPOT assay that’s been adapted to flow cytometry, and ELISPOT assays were being used in the early 1990s.[]
  6. I know it’s debatable, but that’s close enough for a first approximation[]
  7. Walsh, C. M., Matloubian, M., Liu, C. C., Ueda, R., Kurahara, C. G., Christensen, J. L., Huang, M. T., Young, J. D., Ahmed, R., and Clark, W. R. (1994). Immune function in mice lacking the perforin gene. Proc Natl Acad Sci U S A 91, 10854-10858.andKagi, D., Ledermann, B., Burki, K., Seiler, P., Odermatt, B., Olsen, K. J., Podack, E. R., Zinkernagel, R. M., and Hengartner, H. (1994). Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369, 31-37.[]
March 26th, 2008

Redirecting killers

Mouse splenocytes (T cells, B cells, dendritic cells)

Normal mouse spleen: B cells (red), CTL (green), dendritic cells (blue)

We know that HIV can be controlled by an appropriate immune response. Cytotoxic T lymphocytes (CTL) are capable of very effectively suppressing HIV; in fact, in a standard HIV infection, the virus typically spends most of its early phase being controlled by a T cell response. In most people, unfortunately, the control is temporary; since HIV replication is sloppy, the virus throws off mutants at regular intervals, and eventually one of the mutants will be invisible to the dominant CTL response. That mutant replicates rapidly (probably damaging the immune response as it does so) until a new CTL response brings that virus under control, only for other variants to arise again.

Some people are apparently able to hold the virus under control for very long periods — the long-term non-progressor HIV patients. Some of these people seem to have T cell responses against part of the virus that has very precise sequence requirements; if the virus mutates away from CTL recognition, the virus is crippled and can’t replicate effectively. Other people seem to have a broad T cell response, one that recognizes several parts of the virus at once. The odds of successfully mutating all of the targeted areas simultaneously are exponentially lower than of mutating a single region.

Obviously, either of these are states that vaccine designers want as outcomes. That’s not all that easy. People are variable, and there don’t seem to be general rules that you can use to force an immune response to the target of one’s choice. 1 Wouldn’t it be nice if there was a way of bypassing the whole messy immunization step, and just moving straight on to the desired finale of CTL specific for the target of one’s choice?

A paper in the March ’08 issue of Journal of Virology2 does just that.

When you induce T cell-mediated immunity, whether through a vaccine or a real infection, what you’re actually doing is expanding a pool of T cells whose receptor recognizes your special antigen. There are a huge number of potential T cell receptors (TcRs); under normal conditions, any particular antigenic target might have only 20 or 100 T cells that can recognize it, scattered among the millions of T cells with irrelevant specificities. Once a T cell finds its antigen, though,3 that T cell clone divided and expands enormously, as much as 100,000 times. The next time that antigen rides through town, it finds hundreds of sheriffs awaiting it, not just one or two.

HIV budding from a T cellIf the TcR is all you need for specific recognition, can you bypass the whole annoying specific recognition and expansion step? Why not take the TcR from a previous clone, that you already know is useful (perhaps one from another individual altogether) and swap it into generic, non-specific T cells? In fact, that’s been done in a number of cases, and it actually seems to work.4

Joseph et al. tried this with a TcR specific for a HIV antigen. They swapped this known TcR into ordinary generic T cells from a normal blood donor, and turned those boring old plain T cells into CTL that specifically killed HIV-infected cells.

OK, their system is very artificial, involving transformed target lines and a Rube Goldbergesque mouse system to test “in vivo activity”, so it’s not really possible to draw any conclusions about clinical potential. In an actual infection, you’d presumably want to do this with multiple TcRs simultaneously, to target many HIV antigens at once and reduce the risk of immune escape (otherwise, just putting in one chimeric TcR is not different from getting a strong CTL response to HIV — which we know is not sufficient in the long run). I don’t think we know what would happen in that situation; would there be competition between the different TcRs to the point that most would be outcompeted and swamped, ending up with a de facto single target after all? 5

Another question I have is whether the original TcRs might cause mischief — if the T cell has two TcRs, stimulation through one might lead to reactivity with the other, and if the other, original, TcR happens to react with a self antigen you might get the mother of all autoimmune diseases. So my guess is that this is mostly a cute idea that will never go anywhere (for HIV; I think it has much more potential in tumor treatment).

Still, it really is a neat concept, and I hope some of my questions get addressed.

  1. There are some approaches that can do this, but they also have drawbacks.[]
  2. Joseph, A., Zheng, J.H., Follenzi, A., DiLorenzo, T., Sango, K., Hyman, J., Chen, K., Piechocka-Trocha, A., Brander, C., Hooijberg, E., Vignali, D.A., Walker, B.D., Goldstein, H. (2008). Lentiviral Vectors Encoding Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T-Cell Receptor Genes Efficiently Convert Peripheral Blood CD8 T Lymphocytes into Cytotoxic T Lymphocytes with Potent In Vitro and In Vivo HIV-1-Specific Inhibitory Activity. Journal of Virology, 82(6), 3078-3089. DOI: 10.1128/JVI.01812-07[]
  3. assuming appropriate conditions for activation and so forth[]
  4. E.g. for tumors; Morgan, R. A., Dudley, M. E., Wunderlich, J. R., Hughes, M. S., Yang, J. C., Sherry, R. M., Royal, R. E., Topalian, S. L., Kammula, U. S., Restifo, N. P., Zheng, Z., Nahvi, A., de Vries, C. R., Rogers-Freezer, L. J., Mavroukakis, S. A., and Rosenberg, S. A. (2006). Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314, 126-129.[]
  5. Some models for immunodominance predict this, in fact[]
March 24th, 2008

Increasing virus virulence

Chick embryo, Wellcome ImagesI’ve observed before that the common belief that viruses evolve toward avirulence is not particularly true. It’s more accurate to say that viruses evolve toward improved transmission. Some viruses are better transmitted if they let their host survive longer, but other viruses have to be virulent in order to spread. The former may evolve toward reduced (though not necessarily loss of) virulence, but the latter would “want” to maintain stable virulence.

What about increasing viral virulence? What could drive that?

There’s at least one fairly well-documented example of that. The increase in virulence is probably because of a change in the virus’s environment that  forces the virus to become more virulent in order to continue to transmit efficiently. Ironically, the environmental change is vaccination.

As far as I know — I want to put this up front, to forestall the vaccine loons — there’s no instance where this has happened with a vaccine used for humans. 1 I’m talking about a chicken vaccine, for Marek’s Disease.

Marek’s Disease Virus (MDV) is an extraordinarily interesting virus. It’s a herpesvirus of chickens; it causes, among other symptoms, tumors. MDV was a relatively minor problem when chicken farming was a backyard industry. When very large, intensive commercial chicken farms arose, the virus was able to sweep through flocks and cause truly enormous losses. The first Marek’s Disease vaccine, introduced in the 1960s, reduced losses by some 99%. (Incidentally, this was the first vaccine ever to prevent cancer.)

But the 99% protection rate didn’t last long. Losses began to creep up once again, as more virulent viruses arose. New vaccines have been introduced a couple times; each time losses dropped, but then once again new and increasingly-virulent viruses arose. Marek’s Disease viruses isolated today are far more virulent than the relatively benign viruses of the 1960s and early 1970s; the original vaccine is essentially useless against them.

Marek's disease virulence; Witter 1997The figure at right2 (click for a larger version) shows the virulence of virus strains isolated over a ten-year period — although there’s a lot of variability, there’s a pretty clear upward trend. (This chart — and all the others I could find — only shows changes relatively late in the story, skipping the interesting periods in the 1970s and early 1980s when the first changes in virulence were noted. I think this is a technical issue of having the appropriate strains available for comparison. However, see: Increased virulence of Marek’s disease virus field isolates. Witter RL. Avian Dis. 1997 Jan-Mar;41(1):149-63. doi:10.1016/j.tvjl.2004.05.009 for a more detailed analysis of MDV strain virulence over the years.)

This evolution is actually very reminiscent of the myxoma/rabbit co-evolution story I’ve talked about, here and here. Australian rabbits have evolved to become much more resistant to myxoma virus than their European cousins. In this case, MDV is more analogous to the rabbits than to myxoma — evolving mechanisms to persist and replicate in the face of a lethal challenge (for the rabbits, myxoma virus; for Marek’s Disease virus, the vaccine-derived immunity).

Before rabbits could evolve resistance, there had to be some survivors of myxoma infection. In that case, myxoma virus itself evolved to become somewhat less virulent (70-90% lethal, instead of 98%). In the Marek’s Disease story, a key factor is that the vaccines all suck3 in their ability to actually prevent infection; they prevent the disease, but viruses can still infect vaccinated birds, although the virus replicates slower (which reduces transmission).

This is a recipe for virulence. Viruses in general evolve toward improved transmission. The MDV vaccine reduces, but doesn’t eliminate, transmission. Increasing replication in the face of the vaccine increases transmission. Increasing viral replication also increases viral virulence.4

This probably isn’t the whole story (there’s some evidence that the virus was already evolving toward increased virulence even before the vaccine was introduced — perhaps related to changes in its environment brought about by factory farming), and the mechanisms underlying the changes in virulence are not known, but the solution would seem to be clear: Develop a Marek’s Disease vaccine that will induce sterilizing immunity, as do most vaccines used against human viruses. That way, there’s no survivor virus that can act as a seed for evolution of virulence.

Unfortunately, of course, herpesviruses like MDV are notoriously difficult to vaccinate against. There’s still no commercial vaccine against herpes simplex virus, in spite of decades of research. Feline herpesvirus vaccine, which is universally used among pet cats, is like Marek’s in that it prevents symptoms but doesn’t prevent infection. (There is an effective vaccine against varicella-zoster virus [chicken pox] which does seem to effectively prevent infection — an exception to the rules.) So the chicken world is forced to stick with the non-sterilizing vaccines, even though “MD vaccines also appear to have a malign influence on the continued evolution of the pathogen itself.” 2

  1. I’m not saying there’s no such instance, but I don’t know of one.[]
  2. Nair, V. (2004). Evolution of Marek’s disease — a paradigm for incessant race between the pathogen and the host. The Veterinary Journal DOI: 10.1016/j.tvjl.2004.05.009[][]
  3. Note rigorous technical terminology[]
  4. This is not a universal equation; virus virulence isn’t necessarily linked to increased replication, for example.[]
March 21st, 2008

Immunity causes cancer (sometimes)

Anatomy, illustrating chronic inflammation (Wellcome Images)
Chronic inflammation

It’s pretty well-established now that the immune system can, and normally does, protect us against cancer. In particular, the adaptive immune response (especially T cells) clearly limits cancer growth, so that the only cancers we can detect clinically are those that have developed defenses against the adaptive immune response (see here for more).It seems paradoxical, then, that the immune response may also help cancers develop. However, there is a fair bit of evidence that a chronic immune response can actually help drive cancer development. This is especially true for the innate immune system, but long-term stimulation of the adaptive immune system may also be carcinogenic.

The role of the innate system is relatively easy to understand (at least, it made sense to me, which is no guarantee that it makes sense). Chronic inflammation is a bad thing — there are lots of checks built in to the immune response to try to prevent that — and conditions where there’s chronic inflammation are often clearly associated with cancer. The example that jumps to my mind is hepatitis B infection. As far as I know, it’s not generally believed that the virus itself is carcinogenic per se (that is, in contrast to things like Kaposi’s Sarcoma herpesvirus, or some human papillomaviruses, which seem to have the ability to drive infected cells into a de-regulated state). Rather, the increased risk of cancer associated with HBV infection (about five to fifteen times higher than the general population) is probably because of the chronic inflammation that the virus infection causes. 1

There are a number of ways the chronic inflammation can lead to cancer. Simply increasing cell turnover (as cells are killed by the inflammation and have to be replaced) increases the chance of a dangerous mutation arising. Inflammatory factors can act as growth factors for tumor cells. Reactive oxygen species produced as part of the inflammation may increase mutation frequency. And so on. 2

Inflammation and angiogenesis are hallmarks of squamous carcinogenesis in HPV16 transgenic mice.
Inflammation in carcinogenesis

It’s a little more surprising to contend that the adaptive immune system may also help drive cancers. My first response to the concept was to dismiss it, because immune-deficient mice actually have more tumors than wild-type mice, not fewer. However, as I realized within ten seconds of my dismissal, that’s not counterevidence; adaptive immunity could drive cancer at one stage and protect against it at another stage, and the experiments in question would only reveal which of the processes had the larger effect. And in fact, it turns out that although immune-suppressed people (AIDS patients, or transplant recipients) have increased risk of many tumors, they are at reduced risk of other kinds. For example, prostate tumors are less frequent in AIDS patients than in matched controls,3 and breast cancers are less common in transplants recipients4

I don’t think the mechanism(s) underlying this are as well understood as for innate immunity (and that itself is still not well understood). It’s likely that adaptive immunity plays a part in establishing some forms of chronic inflammation. In any case, there’s a fair bit of interest in blocking inflammation during cancer as a component of treatment.

It’s worth emphasizing that the great majority of tumors — if they show any change in incidence in immune-suppressed people — are more frequent; it’s just a few types of tumors that are less frequent. Don’t go immune-suppressing yourself in an attempt to avoid cancer.

  1. There are some virus factors that might be more directly correlated with cancer, but the link is rather indirect.[]
  2. Here’s a nice review:de Visser, K.E., Eichten, A., Coussens, L.M. (2006). Paradoxical roles of the immune system during cancer development. Nature Reviews Cancer, 6(1), 24-37. DOI: 10.1038/nrc1782[]
  3. Frisch, M., R. J. Biggar, E. A. Engels, and J. J. Goedert. 2001. Association of cancer with AIDS-related immunosuppression in adults. JAMA 285: 1736-1745.[]
  4. Stewart, T., S. C. Tsai, H. Grayson, R. Henderson, and G. Opelz. 1995. Incidence of de-novo breast cancer in women chronically immunosuppressed after organ transplantation. Lancet 346: 796-798.[]
March 20th, 2008

Tumor Immunology Top Ten List

Dr Camlee's Cancer Specific (Wellcome Images)My 4-year-old son has been sick all week, probably with influenza (his doctor diagnosed it as influenza, but didn’t perform any specific tests) so I’ve got a little behind on my work,1 and today’s post is going to be a little short. I’m intrigued by an article in the latest Immunological Reviews, which I’ll probably post on later today or perhaps tomorrow, depending on how fast my cells are growing; if I need to split them, the blog comes second.

But before I get to that, I want to point out the introduction to the issue, a tumor immunology top ten list by Olivera Finn, which she introduces thus:2

I am often surprised with the unawareness that can be encountered in the scientific and medical community about the great extent of knowledge that has accumulated in tumor immunology.

I’ve talked about a number of the items on her list, so I’m presenting the list both with the references Finn offers and, where possible, my own posts on the subject:

No.10. Tumors express antigens recognized by the immune system; many have been fully characterized.
Graziano DF, Finn OJ. Tumor antigens and tumor antigen discovery. Cancer Treat Res 2005;123:89–111.
I’ve talked about one example of this here

No.9. Tumors are seen as dangerous by the immune system.

Rock KL, Hearn A, Chen CJ, Shi Y. Natural endogenous adjuvants. Springer Semin Immunopathol 2005;26:231–246.
One of my first posts here was about this.

No.8. There is specific and effective immune surveillance of cancer.
Dunn GP, Old LJ, Schreiber RD. The three Es of cancer immunoediting. Annu Rev Immunol 2004;22:329–360.
I have several posts on this, including this one (specifically about the Dunn et al paper, in fact), and others here and here.

No.7. The immune response is an important biomarker in cancer

Finn OJ. Immune response as a biomarker for cancer detection and a lot more. N Engl J Med 2005;353:1288–1290.
I made a rather peripheral mention of this here.

No.6. Immune responses against cancer can be both good and bad
de Visser KE, Eichten A, Coussens LM. Paradoxical roles of the immune system during cancer development. Nat Rev Cancer 2006;6:24–37.
This is the one I want to talk about.

No.5. Tumors fight back but do not always have to win

Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive strategies that are mediated by tumor cells. Annu Rev Immunol 2007;25:267–296.
I’ve talked about immune evasion by tumors in several places: Here and here, for example.

No.4. Passive immunotherapy of cancer is effective

June CH. Adoptive T cell therapy for cancer in the clinic. J Clin Invest 2007;117:1466–1476.
Cheson BD. Monoclonal antibody therapy for B-cell malignancies. Semin Oncol 2006;33 (Suppl.):S2–S14.

No.3. Active immunotherapy of cancer (cancer vaccines) is marginally effective and can be improved
Finn OJ. Cancer vaccines: between the idea and the reality. Nat Rev Immunol 2003;3:630–641.

No.2. Combination of immunotherapy and standard therapy is possible

Emens LA, Jaffee EM. Leveraging the activity of tumor vaccines with cytotoxic chemotherapy. Cancer Res 2005;65:8059–8064.

No.1. Cancer immunoprevention is an attainable goal

Lollini PL, Cavallo F, Nanni P, Forni G. Vaccines for tumour prevention. Nat Rev Cancer 2006;6:204–216. 26.

  1. As the butcher said when he backed into his sausage machine[]
  2. Olivera J. Finn (2008) Tumor immunology top 10 list. Immunological Reviews 222 (1) , 5–8 doi:10.1111/j.1600-065X.2008.00623.x []
March 17th, 2008

Controlled TReg production

Saints Cosmas and Damian performing a miraculous cure by transplantation of a leg/The Master of Los Balbases.I’ve previously posted on regulatory T cells (TRegs) and their potential role in transplants. Briefly, TRegs are capable of specifically shutting off immune responses to particular antigens; they’re normal components of an immune system. TRegs can be damaging in some contexts — for example, in cancer, where it seems that TRegs often shut off immune responses to tumors, so that the tumor can escape immune clearance; and they can be beneficial in other context — for example, in some persistent virus infections, where a chronic immune response would be damaging, TRegs apparently modulate the immune response so that the virus persists but doesn’t cause severe damage.

There are a couple of obvious scenarios where it would be nice to be able to control TRegs. There’s a lot of interest in reducing TReg activity in cancer, such as with CTLA4 antagonists. There’s also a lot of interest in increasing TReg activity in organ transplants, and there have actually been a couple of cases where it’s seemed to have worked.

A recent paper in PNAS1 offers steps toward a more general procedure, that could in theory lead to controlled, planned generation of TRegs for any antigen.

A key aspect of TRegs is that they are antigen-specific. They don’t randomly suppress immune responses; they identify particular antigens that should be tolerated, and shut off immunity to those antigens. That allows fine control over the response, but it also makes it harder to catch a TReg; T cells (not just TRegs) that recognize any particular antigen are very rare events, hiding in a blizzard of other specificities. What if you could force T cells for an antigen you choose to enter the TReg pathway?

Regulatory T cells (J Clin Invest cover)This has already been done, in fact, but in a very artificial system — in mice with transgenic T cell receptors. These mice overwhelmingly express a single TcR in all of their T cells — there’s no snowflake in a blizzard problem, because the entire blizzard is made of identical flakes. Harold von Boehmer’s group has shown that you can drive these transgenic T cells into the TReg pathway by offering very, very low levels of antigen, under defined conditions, over a long period. 2 The recent paper1 shows that you can do the same thing in normal, non-transgenic, mice; and by doing this you can force graft tolerance. (They used female mice and drove tolerance to the male antigen H-Y antigen. The tolerized female mice then became tolerant of male grafts, while the control female mice rejected the male grafts.)

The key, at least for this particular protocol, seems to be to use very low dose antigen and “suboptimal” conditions (where “optimal” refers to conditions that drive conventional immune responses. The vocabulary of immune responses is really kind of misleading, because it’s focused on easily-measured responses like protection against viruses or graft rejection. Regulatory T cell responses are just as active, and probably are just about as common and important, but it’s hard to talk about them without giving the impression that they’re somehow passive, or abnormal, or defective).

One problem with moving this into the clinic is that you would need to know what the target antigen is, which in an outbred population like humans you do not know a priori. However, as bioinformatic and experimental techniques for identifying antigen peptides improve, it may become more practical to run this for patients before their transplants. The potential payoff would be very high, because you might be able to remove immunosuppression altogether:

If a procedure as simple as peptide infusion, which permits de novo induction of Tregs from mature T cells, prevents transplant rejection or GVHD, it could offer a realistic opportunity to induce tolerance to a variety of antigens such as allergens, transplantation antigens, and antigens causing autoimmunity while minimizing undesirable side effects often associated with general immunosuppression.

  1. Verginis, P., McLaughlin, K.A., Wucherpfennig, K.W., von Boehmer, H., Apostolou, I. (2008). Induction of antigen-specific regulatory T cells in wild-type mice: Visualization and targets of suppression. Proceedings of the National Academy of Sciences, 105(9), 3479-3484. DOI: 10.1073/pnas.0800149105[][]
  2. Kretschmer, K., Apostolou, I., Hawiger, D., Khazaie, K., Nussenzweig, M. C., and von Boehmer, H. (2005). Inducing and expanding regulatory T cell populations by foreign antigen. Nat Immunol 6, 1219-1227.
    Apostolou, I., and von Boehmer, H. (2004). In vivo instruction of suppressor commitment in naive T cells. J Exp Med 199, 1401-1408.[]
March 16th, 2008

Does this make any sense?

Leaf-cutter antSome leaf-cutter ant lineages are more likely to become queens than other lineages; they “cheat”. These lineages are a minority, about 20%, of all leaf-cutter lineages. I’m fine with all that. What puzzles me is this quote:

“The rarity of the royal lines is actually an evolutionary strategy by the cheats to escape suppression by the altruistic masses that they exploit.”

Bill Hughes, quoted in Science Daily News. It’s not a misquote, either; the abstract of the paper in question1 says essentially the same thing:

The rarity of royal cheats is best explained as an evolutionary strategy to avoid suppression by cooperative genotypes, the efficiency of which is frequency-dependent.

What am I missing here? The strategy is successful because it’s rare, sure. Is he arguing that there is positive selection for rarity, as opposed to a strategy that is selected for when it’s rare, and selected against when it’s common?

  1. Proc. Natl. Acad. Sci. USA doi:10.1073/pnas.0710262105
    Genetic royal cheats in leaf-cutting ant societies
    William O. H. Hughes, and Jacobus J. Boomsma []
March 13th, 2008

Immune evasion does work

Sand rat, Psammomys obesus
Sand rat, Psammomys obesus

Although a lot of viruses have ways of blocking recognition by T cells and NK cells, there’s not much known about the importance of these mechanisms in actual infections. That’s because the best-studied viruses in this class tend to be highly species-specific. So, for example, we don’t have good animal models for the human herpesviruses human cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, or Kaposi’s sarcoma herpesvirus. Herpes simplex virus does infect mice, but its immune evasion molecule ICP47 doesn’t work well in mice, so we’re no further ahead.1

Immune evasion by all these viruses has been studied pretty extensively in cultured cells, but because they essentially only infect humans we only have circumstantial evidence for a role in vivo. Similarly, porcine, bovine, and equine herpesviruses encode immune evasion molecules, but pigs, cattle, and horses are not very convenient models for basic research either.Another major group of viruses, besides the herpesviruses, that are noted for immune evasion are the adenoviruses. However, only the human (and primate) adenoviruses contain the classical E3gp19k immune evasion molecule. There’s an animal model for human adenoviruses (the cotton rat) but as I pointed out the other day, there’s little evidence for an important function of CTL immune evasion in this model.

Virus Host Family Genome
Mouse CMV Mouse β NC_004065
Rat CMV Rat β NC_002512
Mouse thymic HV Mouse    
MHV68 Mouse γ NC_001826
Field mouse HV Microtus
Sand rat nuclear
inclusion agent
Sand rat    

So what we need are small animal, and preferably lab mouse, models for infection with adenoviruses or herpesviruses that include immune evasion molecules. As far as we know, mouse adenoviruses don’t have T cell or NK cell immune evasion properties. That leaves us with mouse herpesviruses. Of the hundreds of known herpesviruses, six are known to be murid-specific (see the table at right), and three of those infect lab mice. One of those is totally obscure (there’s very little known about mouse thymic herpesvirus), leaving us with mouse cytomegalovirus and mouse herpesvirus 68 (MHV68). I’ve already commented on immune evasion by mouse CMV. The bottom line is that removing all known T cell evasion molecules from MCMV makes almost no difference to infection or latency; the one difference is that the virus persists longer and at higher levels in salivary glands. That may be important in virus transmission, but lacks a little oomph.

MHV–68 exiting an infected cell on actin-dependent plasma membrane protrusions. EGFP–tagged ORF58 is green, gp150 red, co–localization yellow and nuclei blue (Mike Gill).That leaves MHV68, and I’m pleased to say that there is actually some evidence that T cell immune evasion is important for this guy. (I’ve mentioned this in passing earlier, but it deserves its own post.) MHV68 uses a gene “mK3” to attack MHC class I (MHC class I is recognized by cytotoxic T lymphocytes). 2 In 2002, Philip Stevenson and Stacey Efstathiou made a mutant of MHV68 lacking mK3,3 and tested its ability to infect mice:

Stevenson, P., May, J., Smith, X., Marques, S., Adler, H., Koszinowski, U., Simas, J., Efstathiou, S. (2002). K3-mediated evasion of CD8+ T cells aids amplification of a latent γ-herpesvirus. Nature Immunology DOI: 10.1038/ni818

MHV68 latency +/- mK3 (Stevenson et al, 2002)In cultured cells, where there’s no immune system, the mutant virus grows exactly as well as wild-type MHV68. As well (more surprisingly) there was no difference in the initial virus clearance; the mutant virus and the parent were both cleared from the lungs of infected mice at the same rate, and were undetectable after about 13 days. However (finally!) there was a big difference in the amount of latency. The figure to the right shows latent wild-type (left panel) and mutant (right panel) MHV68, the black dots, in spleens of mice infected 13 days previously. What’s more, during the latent phase there was a better immune response to mutant MHV68; mice infected with the mutant virus had about twice as many CTL specific for MHV68.

The association of higher virus-specific CTL frequencies with lower viral loads suggested that CTLs were responsible for the elimination of DeltaK3 viruses during latency amplification.

Eliminating CTL from the mice removed the difference; in the absense of CTL, the mK3 knockout virus established latency just as well as the wild-type. This shows that effects on CTL, and probably not some other unknown function of mK3, are responsible for the difference.

So with the two and a half authentic models of infection (I count the herpes simplex virus one as a half because it’s more contrived than a natural infection) we have immune evasion molecules helping to establish latency (MHV68), helping to reactivate from latency (herpes simplex) and helping with persistence (MCMV) . In no case is there much effect on acute infection.

  1. However, swapping in a different immune evasion molecule, that does work in mice, helps HSV reactivate from latency; see my previous post here. []
  2. mK3 is so named because it’s highly similar to a Kaposi’s sarcoma herpesvirus gene called “K3”; K3 is one of two KSHV genes that target MHC class I, but we don’t know much about KSHV infection. MHV68 probably isn’t a great model for KSHV, in spite of using a similar protein in immune evasion.[]
  3. This virus was still able to down-regulate MHC class I to some extent, though, so there may be still other immune evasion genes in MHV68[]
March 12th, 2008

Baseball and science

“We do not understand the world; the world is billions of times more complicated than our minds. You can make a useful contribution to a discussion if you can figure out specifically what it is you don’t understand and try to work on it.”

–Bill James

March 10th, 2008

Viral T cell evasion in vivo: The vanishing evidence

Cotton rat, Sigmodon hispidusI’ve lost an old friend. Apparently it’s been dead for quite a while, but I just found out about it:1 E3gp19k doesn’t protect against pulmonary inflammation in cotton rats. A moment of silence, please.

The “friend” in question is the paper
Ginsberg, H., S, Lundholm-Beauchamp, U., Horswood, R., L, Pernis, B., Wold, W., S, Chanock, R., M, and Prince, G., A (1989). Role of early region 3 (E3) in pathogenesis of adenovirus disease. Proceedings of the National Academy of Sciences of the United States of America Proc Natl Acad Sci U S A 86, 3823-3827.

I’ve been using it for years to illustrate a particular point. Unfortunately, while grinding through the literature a couple of weeks ago, I discovered that the paper’s main conclusion was undercut in 1994, and then again in 2002. No one else seems to have noticed this either, so at least I have lots of company.

Viruses have immune evasion genes that allow them to escape or resist the immune response; they target all aspects of the immune system, from interferon to antibodies to cytotoxic T lymphocytes (CTL) to natural killer (NK) cells. I’m particularly interested in the class of viral immune evasion molecules that target CTL recognition by blocking MHC class I antigen presentation. (A summary of antigen presentation is here.) However, as I’ve pointed out before, there is surprisingly little evidence that these genes are important in actual infections, as opposed to their effect in cultured cells. This contrasts with the clear and striking evidence that cytokine escape is a critical virulence factor for several viruses.

CTL evasion in vivo
One big stumbling block in analyzing the importance of antigen presentation blockade in viral infection is that the viruses that have developed this approach tend to be highly species-specific: they’re herpesviruses and adenoviruses. (And HIV; same deal as far as species specificity.) Because, for obvious reasons, research has focused on human pathogens, it’s been hard to move cell culture results into in vivo studies. You don’t see volunteers lining up to be infected with mutant herpes simplex virus, and even where lab animals can be infected with one of these viruses (e.g. herpes simplex) the immune evasion may not work in the lab animal, or there may be some other difference in the infection that makes interpretation difficult.

There are a couple of herpesvirus models. Mouse cytomegalovirus and mouse herpesvirus 68 are both natural mouse pathogens with authentic CTL immune evasion systems. In these cases there isn’t a whole lot of effect from the MCMV CTL evasion molecules (reduced virus titer in salivary glands), and there’s a moderate effect from MHV68 CTL evasion (reduced establishment of latency).

Hela cells infected with adenovirusThere are in fact a bunch of animal adenoviruses, but — strangely — none of these seem to have CTL evasion molecules. At any rate, none of the dozen or so whose genomes I’ve looked at share the human E3gp19k protein that’s long been shown to block MHC class I antigen processing. In particular, mouse adenovirus 1 does NOT block antigen presentation, at least as determined in one careful study. And while human adenoviruses will just about infect mice, it’s not a productive infection. The human adenoviruses do express some genes in mice, but they don’t efficiently go all the way through replication. It’s not a good model for the natural infection, and so there was some interest, 25-odd years ago, when an animal model for human adenovirus infection was identified.

This is the cotton rat, Sigmodon hispidus. It turns out that at least some human adenoviruses, including the popular type 5, replicate quite well in cotton rats and establish a pneumonia that is vaguely reminiscent of the human disease.2 The system has never really become very popular, probably because cotton rats are vicious, evil little bastards that are as much wolverine as rodent. You handle them with steel-mesh gloves, and it’s still a sporting proposition as to whether the researcher or the rat draws first blood. Subsequent experiments showed that in fact the disease in mice looked quite similar to that in cotton rats, suggesting that mice were an adequate model after all,3 and there was an audible sigh of relief as researchers went back to peaceful little mice again.

Adenovirus immune evasion in cotton rats
But before the boom was over, Harry Ginsberg’s group tested what happened when you infect cotton rats with adenoviruses with, or without, the CTL evasion gene E3gp19k. The Ad5 E3 region is mostly involved in immune evasion of various kinds — cytokines as well as CTL — and as such it’s non-essential in cultured cells, where there’s no immune system. It’s not unusual for spontaneous E3 deletions to pop up in virus stocks passaged in tissue culture. Ginsberg infected cotton rats with some of these mutants. Overall, the mutants spread and replicated in the rats just as well as wild-type virus did. But there was one big difference. Here’s the money shot (Ginsberg et al, Fig. 2; click for a larger version):

Cotton rat + adenovirus lungs (Ginsberg et al 1989)

These are cotton rat lungs. On the left, infected with wild-type virus; and on the right, infected with a deletion mutant virus lacking E3gp19k. There’s much more infiltrate (inflammation) in the lungs on the right. The obvious explanation, and the one I’ve used for nearly 20 years, is that E3gp19k actually protects the host as much as the virus. In many cases (especially in pneumonia), it’s inflammation that causes the clinical signs of disease. By reducing CTL recognition of infected cells, E3gp19k reduces inflammation and should reduce the amount of disease.

There are some puzzling parts of this story (why E3gp19k, instead of, say, 14.7k, which reduces cytokines and should have a larger effect on inflammation?) and some weaknesses in the paper (they never actually showed that E3gp19k works in cotton rat cells, for example) but overall it was a clean, clear story that made sense. When Lee Babiss wrote Ginsberg’s obituary in 20034 he included this among Ginsberg’s particularly important acheivements:

They also began to investigate the role in pathology of the adenoviral early gene 3 region, and determined that the proteins encoded by the E3 transcripts influenced the host inflammatory response. This observation led the way to the creation of adenoviral gene delivery vectors that could persist in the host cell for long periods of time, thus promoting prolonged transgene expression.

AdenovirusThe problem is that it’s likely not true.

The E3gp19k mutants were naturally-occurring mutants with actual deletions in the E3 region. There’s no obvious reason why this should present a problem, but an obscure paper in 1994 showed that in fact, it is a problem:

Berencsi, K., Uri, A., Valyi-Nagy , T., Valyi-Nagy, I., Meignier, B., Peretz, F.V., Rando, R.F., Plotkin, S.A., Gönczöl, E. (1994). Early region 3-replacement adenovirus recombinants are less pathogenic in cotton rats and mice than early region 3-deleted viruses. Laboratory Investigations, 71(3), 350-358.

I admit that I haven’t read the whole paper yet. It’s only available on paper (how quaint!) and my request to the library for a copy hasn’t been answered yet. Still, the abstract is very clear. The authors compared the deletion mutant with a replacement mutant — still eliminating E3gp19k, but replacing it with an unrelated gene that restores the genome size to normal (actually greater than normal). The replacement mutant doesn’t show the pathology that the deletion mutant does.

An Ad5 recombinant, Ad-human cytomegalovirus glycoprotein B (Ad-HCMV.gB), in which the E3 region is replaced by the full-length gB gene of HCMV and with a genome size exceeding that of Wt-Ad, induced mild histopathologic responses in cotton rat and mouse lungs, comparable with those of Wt-Ad, but less severe than those of Ad5-delta E3. Analysis indicated that neither class I major histocompatibility complex expression on the cell surface nor differential expression of the protective E3-14.7 kilodalton protein underlies the pathologic differences observed in cells infected with Ad5-delta E3 or the Ad-HCMV.gB recombinant. … Pathogenicity and replication of the recombinant viruses inversely correlate with the genomic size.

(My emphasis.) The lung inflammation is a genome size effect, not an E3gp19k effect.

What’s more, this has been reproduced. In 2001, a second group found exactly the same thing using adenovirus type 4 instead of type 5. 5 They didn’t even know about the Berencsi paper, and didn’t make a connection to genome size (they floundered about trying to explain the effect as a function of the inserted genes), but the actual observation was essentially exactly the same: Pathogenicity is related to reduced genome size, but not to loss of E3gp19k.

As found previously for Ad5, deletion of Ad4 E3 genes resulted in increased lung pathology. Surprisingly, insertion of HIV genes into this region significantly restored protection attributed to E3 gene products, diminishing overall pathologic effects to Ad4WT levels (P<= 0.0001).

Ginsberg’s original paper has been cited over 200 times. Berencsi’s has been cited just 13 times, mainly for technical aspects; as far as I can tell none of the citations actually note the critical observation. Patterson’s paper? Only 4 citations (and as I say, they themselves didn’t cite Berencsi either). Fields Virology, the authoritative source, mentions Berencsi et al in passing but doesn’t describe or comment on the actual finding, let alone its significance, and doesn’t mention Patterson et al at all. Fields offers the party line, Ginsberg’s interpretation, on pulmonary inflammation.

It’s possible that people in the field are aware of the observation and are discounting it for some good reason, but if so, it’s apparently an unpublished good reason. (Maybe when I see the paper I’ll decide it’s a load of dingo’s kidneys, but it’s hard to see how it could go that wrong; what’s more, the replication by Patterson et al make it much more likely that the phenomenon is real.) If it’s not E3gp19k deletion that’s causing the causing the inflammation, what is it? I have no idea. Perhaps the deletion alters regulation or expression of another gene (perhaps by altering splicing); perhaps it alters the rate of genome replication; perhaps (because this is 2008) there’s some microRNA effect. Who knows? The important thing is now I have one less piece of evidence that CTL evasion is important in vivo.

  1. And apparently no one else has realized it yet[]
  2. Pacini, D. L., Dubovi, E. J., and Clyde, W. A. J. (1984). A new animal model for human respiratory tract disease due to adenovirus. J Infect Dis 150, 92-97.[]
  3. Ginsberg, H. S., Horswood, R. L., Chanock, R. M., and Prince, G. A. (1990). Role of early genes in pathogenesis of adenovirus pneumonia. Proc Natl Acad Sci U S A 87, 6191-6195.
    Ginsberg, H. S., Moldawer, L. L., Sehgal, P. B., Redington, M., Kilian, P. L., Chanock, R. M., and Prince, G. A. (1991). A mouse model for investigating the molecular pathogenesis of adenovirus pneumonia. Proc Natl Acad Sci U S A 88, 1651-1655.[]
  4. Babiss, L. E. (2003). In memoriam: Harold S. Ginsberg (1917-2003). Arch Virol 148, 1655-1657.[]
  5. Patterson, L. J., Prince, G. A., Richardson, E., Alvord, W. G., Kalyan, N., and Robert-Guroff, M. (2002). Insertion of HIV-1 genes into Ad4DeltaE3 vector abrogates increased pathogenesis in cotton rats due to E3 deletion. Virology 292, 107-113.[]