Mystery Rays from Outer Space

Smallpox pustules (R. Carswell, 1831)

In contrast to the many viruses that block antigen presentation by MHC class I, only a handful appear to block presentation by MHC class II.  I don’t understand why any would try to block MHC class II in the first place, but another example of it has just been published.

A little background: Major histocompatibility complexes (MHC) are recognized by T cells. T cells come in several flavors, the best-understood of which are CD4 (T Helper) and CD8 (cytotoxic T lymphocyte; CTL) lymphocytes. CD8 T cells are fairly specialized to deal with cells infected with viruses;¹ they recognize MHC class I. CD4 T cells are at the top of the adaptive immune response; they coordinate subsequent responses, by calling in other cell types, driving antibody or CTL responses, and so on.

MHC class I is on the surface of most cells, as you’d expect, because most cells can be infected with viruses. MHC class I is, among other things, a way of directing the CTL attack to the appropriate, virus-infected, cell, and so they deal, fairly strictly, with what’s going on inside their own particular cell. They don’t take up proteins from outside the cell, because then the cell might get killed when it’s actually a neighbor that’s infected. ²

MHC class II, on the other hand, is a general alarm call that signals “Something’s invading the body, somewhere”. MHC class II is only on a limited number of cells, but those cells do take up protein from outside themselves and show it to CD4 T cells. Presentation on MHC class II does not mean that the particular cell is infected.

So it’s quite logical that viruses would be interested in blocking MHC class I, and as I say there are now many examples of viruses that do so. It’s also logical for viruses to want to block MHC class II, since doing so would reduce all the immune responses against them — antibodies, T cells, whatever.

But how would that work? Again: The cells that do MHC class II antigen presentation are not necessarily infected cells. If a virus is going to block MHC class II, it would have to go out of its way infect the MHC class II-presenting cells (known as professional antigen-presenting cells; APC). Not only that, it would probably have to infect a lot of them, to make a real impact on the overall CD4 T cell response, because even a few unaffected APC will drive a fairly significant immune response, making the suppressed ones irrelevant.

So even though viruses might “want” to block MHC class II, there are practical problems that make it hard to do. Nevertheless, there are a couple of viruses who have genes that can block MHC class II. Human cytomegalovirus is the clearest example, I think,³ and several groups have shown that vaccinia virus blocks MHC class II presentation in infected cells.⁴ Now a paper in Virology⁵ argues that the vaccinia gene catchily called “A35″ is responsible for this block. Since close relatives of A35 are present in many other poxviruses, MHC class II blockade may be widespread in this family.

Colocalization between A35 and RhoB in endosomes5

The data are reasonably convincing, though there are some complications. ⁶ But I’m still puzzled by how this is supposed to work. Vaccinia virus, and poxviruses in general, aren’t renowned for infecting dendritic cells and macrophages, which are the cell types they’d have to efficiently target if MHC class II blockade was to help them.

Removing A35 from vaccinia makes it much less virulent in mice:

A mutant A35 deletion virus (A35?) replicated normally in several tissue culture cell lines, but was highly attenuated (100–1000 fold) in the intranasal and intraperitoneal mouse challenge models⁷

And apparently this is associated with a reduced immune response to the virus:

Thus far our animal model data are consistent with this hypothesis, showing a reduction in both VV specific antibody and splenic T lymphocyte responses. ⁸

Which is consistent with a blockade of MHC class II, true, but if you have reduced viral replication for any reason you’d also expect reduced immune responses, because there would be less viral antigen to drive the response. That is, even though A35 blocks MHC class II, and A35 increases virulence, I’m not convinced that A35 increases virulence because it blocks MHC class II. Viral proteins are notoriously multifunctional, and I wonder if the MHC class II blockade is just one function of A35; or perhaps even if it’s just a side-effect of the “real” virulence function.

I’m open to the notion that A35 (and other viral proteins) are true MHC class II blockers, and that this is functionally important, but I’d like to see more data before I put it in the bank.

1. Also, intracellular bacteria, intracellular parasites, and tumor cells[↩]
2. There are exceptions to this rule, including an important phenomenon called “cross-priming” or “cross-presentation”, but that’s not relevant to this discussion now.[↩]
3. For example, Johnson DC, Hegde NR. Inhibition of the MHC class II antigen presentation pathway by human cytomegalovirus. Curr Top Microbiol Immunol. 2002;269:101-15.[↩]
4. For example, Li, P., Wang, N., Zhou, D., Yee, C.S., Chang, C.H., Brutkiewicz, R.R., Blum, J.S., 2005. Disruption of MHC class II-restricted antigen presentation by Vaccinia virus. J. Immunol. 175 (10), 6481–6488.[↩]
5. Rehm, K., Connor, R., Jones, G., Yimbu, K., & Roper, R. (2009). Vaccinia virus A35R inhibits MHC class II antigen presentation Virology DOI: 10.1016/j.virol.2009.11.008[↩][↩]
6. For example, it looks as if there may be other genes, besides A35, that also contribute to MHC class II blockade.[↩]
7. Roper, R.L., 2006. Characterization of the Vaccinia virus A35R protein and its role in virulence. J. Virol. 80 (1), 306–313.[↩]
8. Rehm, K.E., Jones, G.J.B., Tripp, A.A., Metcalf, M.W., and Roper, R.L., in press. The Poxvirus A35 Protein is an Immunoregulator. J. Virol.[↩]

TRegs infiltrate into a tumor

One of the reasons the immune system doesn’t destroy tumors is the presence of regulatory T cells (TRegs) that actively shut down the anti-tumor response.  For once, there’s a little bit of encouraging news on that front.

TRegs are normal parts of the immune system.  They actively prevent other T cells (and so on) from attacking their target. ¹  What’s more, TRegs are antigen-specific.  That is, they recognize a specific target, just as do other T cells, but instead of responding by, say, destroying the cells (like  cytotoxic T lymphocyte) or by releasing interferon (like a T helper cell) a TReg’s response to antigen is to prevent other T cells from doing anything in response to that antigen.  In other words, TRegs cause an antigen-specific inhibition of the conventional immune response. ²

Back to tumors.  We know that immune responses don’t routinely eliminate tumors by the time they’re detectable.  There is some evidence that lots of very small, proto-tumors, are in fact destroyed by the immune system very early on, before they’re clinically detectable, but those tumors that survive that attack seem to be pretty resistant to immune control.  At least part of that resistance is because TRegs get co-opted into the tumor’s control (see here, and references therein, for more on that).

So if TRegs are antigen-specific, and TRegs control immune responses to the tumor, what are the tumor antigens that are driving the TRegs?

I would have assumed that TRegs are looking at many, many tumor antigens, including both normal self antigens³ as well as classical tumor antigens.⁴  But a recent paper⁵ suggests, to my surprise, that this assumption is wrong.  Instead, “Tregs in tumor patients were highly specific for a distinct set of only a few tumor antigens“. ⁵ What’s more, eliminating TRegs cranked up the functional immune response, but only to those antigens TRegs recognized — as you’d expect, if the suppression is indeed antigen specific.

This is interesting for several reasons.  If TRegs can be specific for tumor antigens, then at least in theory ((In practice, we don’t quite have the tools yet, I think) it should be possible to turn off these TRegs while leaving the bulk of TRegs intact (and therefore not precipitating violent autoimmunity).  It also suggests that if the TRegs are only suppressing a subset of effector T cells, there’s something else preventing most effector T cells from, well, effecting.  Maybe those are antigen non-specific TRegs, or maybe there’s something else we need to know about.

I’d like to see this sort of study replicated, and a little more fine-tuning on identifying the TReg’s targets (the readout was intentionally fairly coarse here, in order to identify as many as possible).  Still, it’s an unexpected, and potentially very useful, observation.

1. It’s still not quite clear how they do this[↩]
2. There are also antigen-nonspecific TRegs, but we will ignore them for now.  They’re not as effective as the antigen-specific sort, anyway.[↩]
3. Because TRegs, unlike most immune cells, can be stimulated by normal self antigens[↩]
4. That is, antigens that are mutated, or dysregulated, and that therefore act as standard targets for immune cells[↩]
5. Bonertz, A., Weitz, J., Pietsch, D., Rahbari, N., Schlude, C., Ge, Y., Juenger, S., Vlodavsky, I., Khazaie, K., Jaeger, D., Reissfelder, C., Antolovic, D., Aigner, M., Koch, M., & Beckhove, P. (2009). Antigen-specific Tregs control T cell responses against a limited repertoire of tumor antigens in patients with colorectal carcinoma Journal of Clinical Investigation DOI: 10.1172/JCI39608[↩][↩]

Our deepening knowledge of the immune evasion mechanisms of malaria is revealing the parasite’s ability to orchestrate the human immune response. … It would thus seem futile to test novel antigens or vaccine platforms without first incorporating features designed to circumvent parasite immune evasion strategies. … The prominent feature of a successful vaccine targeting chronic infectious agents such as malaria may therefore not be the antigens it includes, but rather the strategy used to free the immune system from its shackles.

Casares, S., & Richie, T. (2009). Immune evasion by malaria parasites: a challenge for vaccine development Current Opinion in Immunology, 21 (3), 321-330 DOI: 10.1016/j.coi.2009.05.015

“How to avoid influenza: Gargle Daily”

Every virus that infects a vertebrate, has to be able to deal with the vertebrate immune system. The virus’s ancestors that infected vertebrates must have been able to deal with the vertebrate immune system. Those viruses that couldn’t handle an immune response are extinct.

Some of the ways viruses handle immunity, we don’t think of as really “specific”. Rapid replication, for example, has benefits for the virus that extend past just beating the immune system to the punch. But just about every virus, even the smallest ones, also have some form of specific immune evasion gene — some way of blocking, dodging, diverting, or confusing the immune system.

In spite of this nearly universal presence, we don’t really have a good grasp of precisely what viral immune evasion genes do, as far as supporting viral pathogenesis. (For that matter, it’s only for a handful of viruses that we really have much understanding of the pathogenesis in general.) Some viruses have a huge number of genes that are clearly immune evasion genes, others apparently only have one or two. Sometimes you can knock out an immune evasion gene and virtually destroy the virus’s ability to infect; sometimes the knockout only has a modest effect; sometimes there’s no effect at all, or it may even make the virus more, rather than less, virulent.

Viruses are so different from each other¹ that there are probably few if any general rules for immune evasion. Still, we’re not even at a point yet where we have non-general rules, so the more we learn the more likely we are to see patterns.

Physicians expressing their thanks to influenza. Coloured etching attributed to Temple West, 1803.

Influenza, of course, has its own set of immune evasion genes. The most important one is the NS1 gene.² NS1 blocks the interferon pathway, and to the extent that we can generalize, it seems that blocking interferon is one of the most critical things any virus can do. Almost every virus has some way of meddling with the interferon pathways, whether by preventing interferon from being triggered or inducing resistance to the effects of interferon. It’s been known for quite a while that NS1 does this — prevents interferon from being turned on — for influenza viruses, and it’s also been known that NS1 is very, very important to the virus. Mutant influenza viruses without NS1 are much, much less virulent than wild-type virus, and even targeting NS1 after an infection has started can help treat influenza.

(A flip side of this is that influenza viruses with a particularly effective NS1 may be more virulent. The 1918 pandemic influenza, which had a very high mortality rate,³ seems to have a particularly effective NS1 that can block interferon in several ways, and it’s been shown that swapping just the NS1 from the 1918 virus can make otherwise mild flu viruses more virulent. See my previous post about that.)

But there’s a bit of a paradox here. We know that NS1, the interferon blocker, is important to influenza virus. But we also know that interferon is very important in controlling influenza virus infections. For example, mice that can’t respond to interferons are much more susceptible to infection with avian influenza.⁴ So if NS1 works by blocking interferon, why does interferon still protect?

For that matter, one of the major explanations for why some influenza viruses (like avian flu and the 1918 flu) are so virulent, is the “cytokine storm” hypothesis.  (I talked about cytokine storms here and here.)  According to this concept, these viruses are especially lethal because they induce a huge release of cytokines, such as interferon. Yet at the same time the argument is made that these viruses are the ones with especially effective interferon blockers. If they’re really good at blocking interferon, then why do people die of having too much interferon?

It turns out that part of the answer may be timing. A recent paper from Thomas Moran’s group⁵ shows that in the very earliest stages of influenza virus infection, interferons are not being produced; then, a couple of days in, there’s a sudden big bang of cytokines. Knocking NS1 out of the virus changed this; interferons were produced from the beginning of the infection, and the virus was shut down. They call this phenomenon “stealth replication”:

Our data demonstrate that the initiation of lung inflammation does not begin until almost 2 full days after infection, when virus replication reaches its apex. The migration of lung DCs to lymph nodes and the subsequent priming of naive T cells are similarly subject to this delay. We demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza NS1 protein. We propose that the virally encoded NS1 protein establishes a time-limited “stealth phase” during which the replicating influenza virus remains undetected, thus preventing the immediate initiation of innate and adaptive immunity. ⁵

They point out that in normal human influenza virus infection, symptoms take a couple of days to kick in, which fits because most of the “flu-like symptoms” we talk about are generic effects of cytokines. They also point out that a lot of virus transmission occurs before symptoms — i.e. in the first couple days of infection.

Thus, a stealth phase may also occur in humans and probably functions to maximize the probability of transmission before cytokines such as type I IFNs hamper the normal replicative cycle of influenza virus.⁵

This also helps make sense of the cytokine storm concept, I think. If avian or 1918 NS1 is especially good at preventing cytokines, then there might be a slightly longer stealth period, during which time the virus can replicate more. Then, when the immune system suddenly does become aware of an infection, there’s a huge amount of virus present, and the cytokine response would be correspondingly huge.

We might even be able to generalize to other viruses:

The stealth phase concept is not only applicable to influenza virus but can probably be extended to virtually all “real” human viral pathogens that have been shown to have an asymptomatic incubation time. For example, measles and varicella zoster viruses have a substantially prolonged evasion period that can last up to 2 wk. During this asymptomatic phase, these viruses also transmit to other susceptible hosts. Research aimed at interfering with the stealth phase may lead to the development of novel modulators as preventive treatments that target this early immune evasion mechanism. ⁵

I want to point to a previous post I made here, too, about herpes simplex virus. HSV has a wide range of immune evasion molecules, and we don’t have much understanding of what these things do in a natural infection.Frank Carbone’s group  did experiments with mouse infection that showed that HSV has a very narrow window (less than 24 hours) during which it can move from its original site of infection, in the skin, to neurons where it sets up a life-long infection. If the immune response can control HSV in this window, the virus can’t get into neurons and its life cycle is cut short. I speculated at the time that this might help explain immune evasion by HSV — it wouldn’t have to be super efficient, just keep things under control during that brief, early window. Seems quite similar to the influenza situation: Timing is critical, and perhaps immune evasion is one reason why.

1. “Virus” isn’t a natural division; it groups together things with very different, and completely unconnected, evolutionary histories[↩]
2. “NS” stands for “Non-structural”, meaning that the protein isn’t part of the virion that floats around and infects new cells — rather, the NS1 protein is produced anew in each cell after infection.[↩]
3. As influenza infections go — not close to something like smallpox or ebola, but some 20 times higher than normal seasonal flu[↩]
4. Szretter, K., Gangappa, S., Belser, J., Zeng, H., Chen, H., Matsuoka, Y., Sambhara, S., Swayne, D., Tumpey, T., & Katz, J. (2009). Early Control of H5N1 Influenza Virus Replication by the Type I Interferon Response in Mice Journal of Virology, 83 (11), 5825-5834 DOI: 10.1128/JVI.02144-08[↩]
5. Moltedo, B., Lopez, C., Pazos, M., Becker, M., Hermesh, T., & Moran, T. (2009). Cutting Edge: Stealth Influenza Virus Replication Precedes the Initiation of Adaptive Immunity The Journal of Immunology, 183 (6), 3569-3573 DOI: 10.4049/jimmunol.0900091[↩][↩][↩][↩]

Macrophage phagocytosing mycobacteria

Sometimes the simple, obvious answer is right, and sometimes it’s completely backwards.

Tuberculosis was a terrifying, ubiquitous killer in the 19th century, but is relatively rare today (at least, in developed countries). The reason for the drop in Tb deaths isn’t entirely clear; it started with social factors probably including accidental or deliberate isolation of Tb patients, antibiotic treatment also knocked the disease back, and in some areas the vaccine (known as BCG) made a difference as well.

BCG is one of the oldest vaccines still in wide use; it was developed in the 1920s when a strain of Mycobacterium bovis (tuberculosis of cattle, contagious to humans) spontaneously lost virulence in culture. This avirulent strain of the bacterium was sent around the world and cultured independently, resulting in many distinct vaccine strains in different places and times. These strains are not only distinct genetically, but also phenotypically — they look different in culture, or grow differently, or whatever.

Over time, the vaccine has changed functionally, as well. Very early on the vaccine abruptly became even less virulent. More gradually, it seems that BCG has also become less effective; it’s no longer is able to protect against pulmonary Tb (although it’s still protective against other forms of the disease). Why is this?

At first glance this seems unsurprising. The bacterium has been grown in culture — outside of any animal host — for nearly 100 years. It’s had no selection to maintain its ability to grow in animals, or to avoid their immune responses, so of course it’s going to lose its ability to grow in animals.

But a recent paper¹ suggests that exactly the opposite happened. Whether randomly, or because of some unexpected type of selection, the BCG strain has actually amplified an immune evasion function. This modern variant of the vaccine strain isn’t simply passively failing to induce an immune response; it’s actively suppressing the immune response.

Specifically, the authors argue that normal (wild, virulent) Mycobacterium secretes antioxidants as an immune evasion mechanism; that modern BCG also secretes lots of antioxidants; and that this is related to genomic duplications in some BCG strains:

Some BCG daughter strains exhibit genomic duplication of sigH, trxC (thioredoxin), trxB2 (thioredoxin reductase), whiB1, whiB7, and lpdA (Rv3303c) as well as increased expression of genes encoding other antioxidants including SodA, thiol peroxidase, alkylhydroperoxidases C and D, and other members of the whiB family of thioredoxin-like protein disulfide reductases.¹

In other words, the long-term culture of BCG has yielded variants that are less immunogenic, because they are more actively suppressing the immune response. If their reasoning is correct, then reducing the antioxidant secretion from BCG should increase its immunogenicity. They took a BCG strain and deleted the duplicated antioxidant gene sigH (as well as the overexpressed SodA), and sure enough, the deleted version was more immunogenic and more protective in mice. “By reducing antioxidant activity and secretion in BCG to yield 3dBCG, we unmasked immune responses during vaccination with 3dBCG that were suppressed by the parent BCG vaccine.”¹

As a possible explanation, they note that their deletion variant also grows more slowly in culture than the “wild-type” BCG, and especially under certain culture conditions, and that this has led, coincidentally, to the reduced immunogenicity:

The practice of growing BCG aerobically with detergents to prevent clumping may have increased oxidant stress to cell wall structures and selected for increased antioxidant production. Then with each transfer the bacilli making more antioxidants represented a slightly greater proportion of the culture until they became dominant. In vivo, these mutations caused the vaccine to become less potent in activating host immunity. In effect, we believe that as BCG evolved it yielded daughter strains with an increased capacity for suppressing host immune responses. ¹

If this turns out to be generally true, then there’s a relatively straightforward handle for converting BCG back into a more effective, and safer, vaccine; whereas if the reduced immunogenicity was because of over-attenuation, it’s not so simple — you’d be trying to make a vaccine more virulent, which is a tricky tightrope to walk.

Incidentally, I frequently complain about the terrible, terrible quality of press releases about scientific advances  (and therefore the terrible quality of much “science reporting”, which is basically regurgitating the terrible press releases) so I want to give props to the person at Vanderbilt University Medical Center who put together the release for this paper — it’s a clear, simple, interesting, and as far as I can tell accurate account of the finding, background, and observation.  It can be done well — I wish it was done this well more often.

1. Sadagopal, S., Braunstein, M., Hager, C., Wei, J., Daniel, A., Bochan, M., Crozier, I., Smith, N., Gates, H., Barnett, L., Van Kaer, L., Price, J., Blackwell, T., Kalams, S., & Kernodle, D. (2009). Reducing the Activity and Secretion of Microbial Antioxidants Enhances the Immunogenicity of BCG PLoS ONE, 4 (5) DOI: 10.1371/journal.pone.0005531[↩][↩][↩][↩]

Researchers have used the mouse extensively as a model organism to study the pathogenesis of human infections and found that it imperfectly recapitulates many aspects of infectious disease as seen in patients. ¹

Humanizing a mouse

That strikes a chord with me because I just sent off a grant application explaining that mice are not suitable models for viral immune evasion.  However, my application may show a failure of imagination (or courage), because what Coers et al.¹ are driving toward is humanizing mice to make them better models for human disease, whereas I am merely proposing a different animal model.

What causes species specificity in pathogens?  That is, why is it that many pathogens infect humans very nicely, but don’t infect mice to any extent?  (And, of course, conversely, why do other pathogens cause disease in mice and not in humans.)

Chlamydia trachomatis in human cells

In some cases, a viral pathogen may simply be unable to get into the appropriate cell in the wrong species. An example is poliovirus, which normally doesn’t infect mice at all. But if you make a transgenic mouse² that expresses the (human) poliovirus receptor³, then the virus infects mice, and causes disease in them, perfectly well.   In this case, the receptor is the critical determinant of species specificity.  As a natural example of the same concept, SARS virus at least partly adapted to infecting humans by modifying its receptor-binding protein⁴ to improve interaction with the human version of the protein.

But there are also lots of cases where the virus can get into cells from the other species, yet doesn’t manage to replicate well or cause disease.  I’ve talked about mouse cytomegalovirus (MCMV) and its inability to infect humans here; it turned out that MCMV can’t infect human cells well because its normal ability to disarm the programmed cell death (apoptosis) pathways only works against the mouse versions of the pathway.  There are similar stories with HIV and its primate-infecting cousins; these viruses are limited to infecting hosts in which they (the viruses) can eliminate the APOBEC retrovirus-destroying proteins.  And the poxvirus myxomavirus is at least partly restricted to infecting rabbits because it can only inactivate the interferon pathway in rabbit cells. ⁵

You may notice two things about these examples: First, the non-receptor examples are generally immune evasion stories.  That is, these viruses are often apparently restricted to infecting a limited number of species because their immune evasion arsenal is limited to those species; take away their immune evasion by putting them in the wrong species, and they’re enfeebled.  Second, these examples are viruses.  The reason for that is just that I’m used to dealing with the crisp, clean mountain air of virology, and I don’t usually descend into the fetid swamps of bacteriology.⁶

But it turns out that at least in some cases the principles seem to be the same.  The Coers et al. paper¹ I cited at the top here makes some very familiar points: The receptor half of the story (“Colonization often relies on species-specific interactions of microbial ligands with host cell receptors“) applies to some bacterial pathogens (“Transgenic mice expressing human E-caherin in the small intestine, on the other hand, are susceptible to oral infections with L. monocytogenes and develop enteropathogenicity and systemic infections“).  And the immune evasion half also applies to some bacterial pathogens (“Additionally, host restriction may be caused by the failure of pathogens to deter immune assaults in the non-typical host“).

Even the nature of the immune evasion targets is familiar. Interferon pathways are frequent targets of bacterial immune evasion, as they are of viral immune evasion.  The details are different, in that the instances Coers et al. describe target a different branch of the interferon induction pathway, but the pattern is the same:

… the mouse-adapted strain Chlamydia muridarum, but not its close relative C. trachomatis, can specifically evade IRG-mediated⁷ host resistance … The divergent counterimmune mechanisms employed by the human pathogen C. trachomatis and the mouse-adapted pathogen C. muridarum clearly reflect the differences in the IFN? responses of their respective hosts. ¹

They finally discuss the possibilities of “Mus homunculus”, humanized mice, tailored to each pathogen, that would make more authentic models of infectious disease.  “Though the creation of humanized mouse models for infectious disease will require substantial effort and resources, the long-term benefits of these new models would undoubtedly be enormous.” ¹

1. Coers, J., Starnbach, M., & Howard, J. (2009). Modeling Infectious Disease in Mice: Co-Adaptation and the Role of Host-Specific IFN? Responses PLoS Pathogens, 5 (5) DOI: 10.1371/journal.ppat.1000333[↩][↩][↩][↩][↩]
2. Hi, Vincent![↩]
3. Transgenic mice expressing a human poliovirus receptor: A new model for poliomyelitis.
   Ruibao Rena, Frank Costantinib, Edward J. Gorgaczc, James J. Leeb and Vincent R. Racanielloa
   Cell 63:353-362 (1990) [↩]
4. Li W, Zhang C, Sui J, Kuhn JH, Moore MJ, et al. (April 20, 2005) Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2. EMBO J 24.:1634-43.
   Sheahan T, Rockx B, Donaldson E, Sims A, Pickles R, et al. (March 2008) Mechanisms of zoonotic severe acute respiratory syndrome coronavirus host range expansion in human airway epithelium. J Virol 82.:2274-85.[↩]
5. Wang F , Ma Y , Barrett JW , Gao X , Loh J , Barton E , Virgin HW , McFadden G (2004) Disruption of Erk-dependent type I interferon induction breaks the myxoma virus species barrier. Nat Immunol 5: 1266-1274[↩]
6. In other words, I don’t know much about bacteriology.[↩]
7. IRG is part of an interferon-induction pathway[↩]

Simulation of hA3G-mediated HIV-1 evolution.

One of the host defenses against HIV¹ is “APOBEC3G” and related proteins.  These proteins force HIV to hypermutate, killing its ability to replicate in the next cell it infects.  On the other hand, it looks as if low-level mutation by APOBEC3G over the decades has driven HIV evolution:

We have found that hA3G activity acting on prior generations of virus has left detectable footprints in the HIV-1 genome. ²

HIV can only infect human cells because it has a defense against APOBECs: the viral protein “vif” causes APOBECs to be destroyed, and the virus is able to replicate without being hypermutated. 

So let’s say we develop an antiviral drug that blocks vif.  APOBECs would drive hypermutation of the virus.  This hypermutation would include the gene encoding vif.  Would this mutation, in a kind of molecular judo, drive rapid evolution of vif, so that it becomes resistant to the drug?

According to some experiments ²  both in cells and in silico, perhaps not:

However, since the predicted effect on resistance to standard antiviral drugs is likely to be small, we propose that concerns over increased resistance mutations should not impede development of HIV-1 Vif as a candidate drug target. ²

I’m not quite convinced this paper was actually modeling the phenomenon they say they’re modeling — is vif that’s shut off by deliberate mutations the same as vif that’s blocked by a (hypothetical) drug? But it’s a useful start, anyway.

1. and other lentiviruses; as well as quite a few other virus types[↩]
2. Patric Jern, Rebecca A. Russell, Vinay K. Pathak, & John M. Coffin (2009). Likely Role of APOBEC3G-Mediated G-to-A Mutations in HIV-1 Evolution and Drug Resistance PLoS Pathogens, 5 (4) DOI: 10.1371/journal.ppat.1000367[↩][↩][↩]

We know that the immune system can destroy tumors. We also know, unfortunately, that by the time we see a tumor, immunity probably won’t destroy the tumor. There are lots of reasons for that. One is that tumors are essentially part of the normal body, so it’s normal for the immune system to ignore them. It looks as if you need to have immunity that’s just right to get rid of a tumor.

Tumors arise from normal self cells,¹ that the immune response has been programmed to ignore. Now, the process of becoming a tumor is not normal, and so tumors are not entirely normal self any more — meaning that there are likely to be some targets in most if not all tumors. But in all but the most reckless tumors the differences between abnormal and normal are relatively small, compared to, say, a virus-infected cell that contains many potential targets.

There’s actually a long list of known tumor antigens; the T-cell tumor peptide database lists many hundreds of them. But most are not truly specific for the tumor.  The’re actually normal self antigens; they’re derived from proteins that are overexpressed in tumors, or that are differentiation antigens or “cancer-germline” antigens that are normally also found in self tissues. What’s more, these normal self antigens are the most interesting tumor antigens, as far as clinical utility is concerned. Mutations can make brand-new, non-self targets for the immune system, but they’re going to be sporadic targets, often unique to individual tumors — not something you can prepare for. The normal antigens, though, are likely to be predictable, common targets; it’s conceivable that tumor vaccines can be prepared in advance.

Human melanoma cell

If these antigens were common (which they are, in some tumor types — like melanoma), and they were good targets for the immune system, then we wouldn’t see much cancer. We do see melanomas quite often, and part of the reason may be that the immune system generally responds quite weakly to these antigens.  Why is that? And, more to the point, how can we make the immune system respond more strongly? A recent paper in the Journal of Experimental Medicine² offers answers for both of these questions.

From work in the past couple of years, we now have decent estimates of how many T cells there are that can react with any particular target. (See here and here for my discussion of the earlier papers.) A reasonably strong immune response to a non-self epitope might originate from maybe 100 or so precursor T cells. There’s a rather wide range of frequency for these precursor cells, say from 20 to 1000; and to some extent, the fewer T cells there are the weaker (the less immunodominant) the immune response.

We expect T cells against normal self targets to be less common, because they should be eliminated as they mature in the thymus. Some may survive, though, and we would count on these survivors to attack the normal (albeit overexpressed, or abnormally present) target in the cancer cells. But just how rare are they?

Rizzuto et al say they’re really rare (this was in mice, by the way); at least ten times less abundant than T cells against non-self antigens.  If you look at the range I gave for “normal” precursors, that could mean there are fewer than 5 or 10 precursors.  If the average is “fewer than five”, then quite possibly some mice have only two, or one, or no precursors.  You can’t have much of a response with no precursors.

So there’s a weak anti-tumor response because there aren’t many T cells in the body that can respond to the normal self targets in the tumor. That’s not really a surprise, but it does raise the question, What if there were more of the T cells? To ask that question, Rizzuto et al. tried transferring more of these precursor T cells into tumor-bearing mice — starting at around the normal level for a precursor to non-self antigen, and going up from there — and then vaccinating with the appropriate target.

The effects were pretty dramatic. With no supplemental T cells (that is, with the natural, very low, level of T cell precursors) the mice all died of the tumor quickly. At the middle of the range, almost all of the mice rejected the tumor. And at the highest levels of transfers? The mice all died again. Having enough T cells to respond was protective, but putting in too many made them useless.

These results identify vaccine-specific CD8+ precursor frequency as a remarkably significant predictor of treatment and side-effect outcome. Paradoxically, above a certain threshold there is an inverse relationship between pmel-1 clonal frequency and vaccine-induced tumor rejection.²

Mouse melanoma cell

(My emphasis) This paradoxical effect is probably because the numerous T cells started to compete with each other so that none of them were properly activated; they only saw effective-looking polyfunctional T cells at the lower transfer levels.

In other words, if you’re going to transfer T cells to try to eliminate a tumor, more is not necessarily better. Quality and quantity are both important factors, and quantity helps determine quality.

One question I have is how this relates to tumor immune evasion. Many tumor types  acquire mutations, as they develop, that block presentation of antigen to T cells. Are these mutations perhaps only partially effective — giving the tumors sufficient protection against the tiny handful of natural precursors they “expect” to deal with, but not against a larger attack after, say, vaccination — or are they more complete, and protective even if the optimal number of T cells are transfered? I’d guess that it would depend on the tumor, but it looks as if it might be a relevant question and it would be nice to have more than a guess.

Our results show that combining lymphodepletion with physiologically relevant numbers of naive tumor-specific CD8+ cells and in vivo administration of an effective vaccine generates a high-quality, antitumor response in mice. This approach requires strikingly low numbers of naive tumor-specific cells, making it a new and truly potent treatment strategy.   ²

1. I’m ignoring here crazy things like the contagious tumors of Tasmanian Devils and dogs[↩]
2. Rizzuto, G., Merghoub, T., Hirschhorn-Cymerman, D., Liu, C., Lesokhin, A., Sahawneh, D., Zhong, H., Panageas, K., Perales, M., Altan-Bonnet, G., Wolchok, J., & Houghton, A. (2009). Self-antigen-specific CD8+ T cell precursor frequency determines the quality of the antitumor immune response Journal of Experimental Medicine, 206 (4), 849-866 DOI: 10.1084/jem.20081382[↩][↩][↩]

Hierarchical clustering of breast carcinomas1

Something that’s puzzled me for years is why the same kinds of tumors tend to have the same kinds of immune evasion mechanisms. And I’m not going to give an answer, just trying to share the confusion a little.

What I mean is this:

It has been demonstrated that human tumors of distinct histology express low or downregulated MHC class I surface antigens … The distinct frequency of MHC class I abnormalities is caused by total HLA class I antigen loss, HLA class I down-regulation as well as loss or down-regulation of HLA class I allo-specificities. However, the frequency and mode of these defects significantly varied between the types of tumors analysed and could be associated in some cases with microsatellite instability. ²

(My emphasis) As I’ve noted here several times (most specifically here) tumors very often evade the immune system as they mature. Cytotoxic T lymphocytes (CTL) can control tumors in the tumors’ eary stages, but by the time we detect a tumor clinically the tumor is almost always resistant to the immune system. They do this in various ways, including inducing regulatory T cells, but also by mutating themselves to make themselves invisible to CTL (and other components of the immune system, but let’s keep it simpler for the moment).

There are a myriad ways for a tumor to become invisible, at the molecular level.  The MHC class I antigen presentation pathway is long and complex, and for any partiuclar tumor there are likely to be many different bottlenecks, points of attack.  Since tumors are all independent events³, so at first, and even second, glance, there’s no obvious reason why tumors of the same type should find a similar approach.  That is, just because two colon carcinomas look the same histologically in two different individuals, there’s no link between them.  ⁴ Why should colon carcinomas avoid CTL using one set of mutations, while, say, breast cancers use a different set of mutations? Yet apparently, that’s what tends to happen; for example:

Mutations or deletions in β2-m were detected in colon carcinoma (21%), melanoma (15%) and other tumors (<5%). So far, no mutations in β2-m have been found in RCC lesions, bladder and laryngeal tumors despite MHC class I loss or downregulation. … haplotype loss was found in head and neck squamous cell carcinoma (HNSCC) with a frequency of 36%, whereas in renal cell carcinoma (RCC) LOH only occurs in approximately 12% of tumor lesions analyzed. ²

If we saw these patterns only with virus-associated cancers, such as cervical carcinomas and even hepatic carcinomas, there would at least be a common link, but these tumors are not (as far as we know) caused by viruses in humans.

Part of the answer may be that the particular oncogenes associated with different tumor types lead to particular transcriptional hot-spots, and being a transcriptional hot-spot makes the region a mutational hot-spot as well, but at least as I understand it that’s not enough to account for the trends.

So why are particular MHC abnormalities linked to tumor type?  Anyone?

1. Turashvili et al. BMC Cancer 2007 7:55   doi:10.1186/1471-2407-7-55[↩]
2. Seliger, B. (2008). Molecular mechanisms of MHC class I abnormalities and APM components in human tumors Cancer Immunology, Immunotherapy, 57 (11), 1719-1726 DOI: 10.1007/s00262-008-0515-4[↩][↩]
3. barring such weird things as canine transmissible venereal tumor and Tasmanian Devil facial tumors; see here for more on those[↩]
4. The comparison is, of course, viruses.  A herpesvirus of chickens, and one of humans, may both use immune evasion mechanisms, but they have a common ancestor even if it’s a couple of hundred million years ago.[↩]

It’s well known that HIV mutates rapidly in infected patients in order to escape from the immune system. The mutations in HIV track with the peptides that bind to MHC class I in any particular patient. When the virus is transmitted to a new patient, though, those mutations don’t help it much, because MHC is so variable between individuals that the new infected person will very likely have a different MHC class I pattern. (In fact, the mutations the virus developed in the first patient, are likely to be actively harmful to the virus.) The virus has to start all over again and discover a new path toward immune escape. Over a long enough time, the virus may be able to slowly accumulate mutations that allow it to escape from the worst of the MHC class I alleles (see here for a possible example), but it’s very difficult, simply because MHC is so diverse.

But MHC class I itself is only the final stage of a longish pathway of antigen presentation — the route by which peptides are produced, modified, transferred into the right location, bind to the right proteins, all that stuff. (If it’s slipped your memory a little, I made a summary page for MHC class I antigen presentation here.) Within that pathway, at least in humans, it’s only the MHC class I heavy chain itself that’s wildly diverse; the other steps are pretty similar between any two individuals. So why doesn’t the virus mutate to avoid one of these monomorphic steps, and then not have to worry about re-mutating all over again after the next transmission?

Putting that less teleologically, why don’t mutations in HIV, that allow it to escape from the monomorphic steps in antigen presentation, persist in each new individual and accumulate within the population? Those mutations should be just as beneficial to the virus in the new infected person as in the original infectee.

Rob de Boer’s group  asked this question recently,¹ and found that

… within hosts, proteasome and TAP escape mutations occur frequently. However, on the population level these escapes do not accumulate … ¹

TAP structure2

(My emphasis) And the reason is the same reason other immune escape mutations don’t easily accumulate in the population: MHC is too diverse. If I follow the argument correctly, because the other components of the system are monomorphic, they have a very broad specificity for peptides, whereas MHC itself has a fine specificity. The virus can’t mutate every possible sequence in its genome that would interact with, say, TAP, because there would be thousands of them. If a mutation that prevents TAP binding does arise in one host, it’s selected because it prevents recognition of a particular MHC class I-binding peptide, and when it moves into a new host that peptide is no longer relevant for immune escape, so it’s not selected any more.

That means that, even taking the whole antigen presentation pathway into account:

The total number of predicted epitope precursors and CTL epitopes in a large population data set of HIV-1 clade B sequences is not decreasing over time. ¹

I am a little cautious about accepting this paper completely, because it’s heavily based on database analysis without a lot of testing; we don’t actually know whether the escape mutations they identify for TAP actually do escape TAP, for example. They make a number of arguments, in passing, for the accuracy of the epitope prediction programs out there; I am slowly backing in to some acceptance of the notion that the predictive programs are getting pretty good, which wasn’t my position a couple of years ago, but I still am not convinced they’re as good as they say here.

But the conclusion is fairly simple and straightforward, and it leads to an interesting suggestion:

… we speculate that only one of the steps in the antigen presentation pathway has to be polymorphic to prevent pathogens from adapting to any step in the pathway. The mechanism functions best when the polymorphy occurs at the most specific step in the pathway, as that increases the fraction of epitope precursors that is not under selection pressure. While in humans it is the MHC class I molecules that are highly polymorphic and specific, other solutions do appear to exist. The TAP molecules of rats are more specific than the human TAP, and have a limited functional polymorphism, and the TAP and MHC genes of chickens are equally polymorphic on the nucleotide level ¹

Chicken MHC is an interesting case, and is very strongly linked to resistance to some pathogens. But the reason for the tight linkage to resistance isn’t really known; there’s no obvious reason at the level of the MHC. It might be interesting to look at TAP as part of the resistance, as well.  I have some chicken stuff in the lab, and I should see if we can test that.

1. Schmid, B., Kesmir, C., & de Boer, R. (2008). The Specificity and Polymorphism of the MHC Class I Prevents the Global Adaptation of HIV-1 to the Monomorphic Proteasome and TAP PLoS ONE, 3 (10) DOI: 10.1371/journal.pone.0003525[↩][↩][↩][↩]
2. Structural arrangement of the transmission interface in the antigen ABC transport complex TAP.
   Oancea G, O’Mara ML, Bennett WF, Tieleman DP, Abele R, Tampé R.
   Proc Natl Acad Sci U S A. 2009 Mar 18  doi: 10.1073/pnas.0811260106[↩]