Eliminating TRegs, in mouse models of cancer, often allows a strong immune response to the tumor. An interesting spin on this was shown in a recent J Immunol paper.¹ It seems that the TRegs don’t generally suppress all the response, they shut down the responses to some targets harder than others:

Our results indicate, therefore, that depletion of Tregs uncovers cryptic responses to Ags that are shared among different tumor cell lines. CT26-specific T cell responses can be elicited by different forms of vaccination in the presence of regulatory cells, but in these cases T cell responses are highly focused on a single tumor-specific epitope …Taken together, these data suggest that immune responses to some Ags are more tightly regulated than others.  ¹

In other words, even though you might be able to force a protective immune response to a tumor by vaccinating in the presence of TRegs, when you get rid of TRegs the response is broader, and targets T cell epitopes that otherwise wouldn’t look like they’re epitopes at all.

I wonder if this goes on with “normal” (say, viral or other non-tumor) epitopes – whether this sort of thing might help explain some forms of immunodominance. I kind of doubt it, but the phenomenon does sounds a little like revealing a subdominant response.

I wonder also how this ties in with a recent paper that suggested TRegs in tumors are highly focused on a small subset of tumor epitopes. Could they be more broadly-based, but on epitopes that are otherwise invisible? Again, I kind of doubt it, but it’s an intriguing idea.  Maybe the universe of tumor epitopes available for attack is much larger than we realize.

1. James, E., Yeh, A., King, C., Korangy, F., Bailey, I., Boulanger, D., Van den Eynde, B., Murray, N., & Elliott, T. (2010). Differential Suppression of Tumor-Specific CD8+ T Cells by Regulatory T Cells The Journal of Immunology, 185 (9), 5048-5055 DOI: 10.4049/jimmunol.1000134

We know that lots of viruses, especially herpesviruses, block antigen presentation. The assumption has been that they are thereby preventing T cells from recognizing infected cells, though long-term readers of this blog¹ will know that I’ve been puzzled about the details of this for quite a while.

A recent paper² raises yet another complication for this pathway: In humans³ there are T cells that specifically recognize cells in which antigen presentation is blocked:

Our data indicate that the human CD8+ T cell pool comprises a diverse reactivity to target cells with impairments in the intracellular processing pathway²

If so, you might wonder why the viruses would bother blocking antigen presentation. They might avoid recognition by T cells specific for the viral proteins, but at the cost of being recognized and eliminated by the T cells that recognize antigen-presentation-defective cells.

As always, I don’t have an answer. I do have the unhelpful observation that viruses are incredibly subtle and efficient, and given that herpesviruses have apparently maintained the ability to block antigen presentation for some 400 million years it’s presumably useful to them. I’ll also add the even more unhelpful observation that immune systems are also incredibly subtle and efficient and have also persisted for 450 million years.

However, there may be a clue in the techniques that Lampen et al used to turn up this subset of T cells: They used multiple rounds of stimulation, which is going to expand these cells massively. We don’t know how abundant they are inside a normal human – perhaps they are so rare that they don’t have a chance to impinge on herpesvirus infection early enough.

The catch with that, though, is that tumors also frequently get rid of antigen presentation via mutation; in fact, eliminating antigen presentation seems to be one of the most common forms of mutations in cancers, suggesting that it’s an important part of their ability to survive and expand in the face of immune attack. Tumors are immunologically present much longer than viruses ((Although herpesviruses set up a lifelong infection, most of that is generally in a non-immunogenic, latent form). So why doesn’t this long-term tumor presence lead to amplification of these antigen-presentation-deficient-specific T cells that would eliminate the tumor?

My guess here is that this is where TRegs come in. As I said in a recent post, TRegs are very commonly, if not universally, associated with tumors, and prevent immune attack on the tumor. I wonder if the tumors mutate to avoid T cell recognition early in their development, before they are able to trigger the TReg response; that allows them to grow large enough and long enough that by the time the presentation-defect-destroyers kick in, the tumors have their TReg defenders set up.  (I admit that this doesn’t account for the correlation between a tumor’s loss of antigen presentation, and poor prognosis, but I leave this as an exercise for the reader.)

And, of course, where either of these defense systems for the proto-tumor fails, we normally would simply not see any tumor at all. Perhaps this is happening all the time inside us — proto-tumors are being eliminated by T cells, some are mutating their antigen presentation pathway and lasting a little longer and are then eliminated by a different subset of T cells, and we never know it.

1. If any
2. Lampen, M., Verweij, M., Querido, B., van der Burg, S., Wiertz, E., & van Hall, T. (2010). CD8+ T Cell Responses against TAP-Inhibited Cells Are Readily Detected in the Human Population The Journal of Immunology DOI: 10.4049/jimmunol.1001774
3. As has been previously shown in mice

There’s increasing evidence supporting the notion that tumors are often not rejected by the immune system because regulatory T cells actively block the immune response to the tumor cells. ¹

That means that within the tumor, two branches of the immune response are fighting it out. If the TRegs win, the tumor will not be rejected (and may eventually kill the host); if the rejection branch² wins, the tumor may be rejected and the host may survive a little longer.

Both TRegs and rejection-branch T cells are driven by specific antigen. That is, as opposed to the general patterns that drive innate immune responses, the T cells are activated by peptides associated with major histocompatibility complexes (mainly class II MHC, for the TRegs).

So that raises an interesting question: What specific peptides activate the TRegs in the tumors, and are they different from the ones that activate rejection-type CD4s?

The question is even more interesting than it may seem at first glance, because³ there are different TReg subsets with different peptide preferences. One set of TRegs likes to see ordinary self-peptides: Peptides that are naturally present, and that should not be rejected because, well, they’re part of you. “Normal” rejection-type T cells don’t see those peptides, because those that do are killed during their development (or are converted into TRegs during development, probably). The other group of TRegs sees foreign peptides, that would be expected to be rejected. You need these TRegs as well, because there are times when a chronic immune response, even to a foreign invader, is more harmful than the invader itself; so under those circumstances, some rejection-type T cells get converted into TRegs, and those can shut down the response to the invader, hopefully to reach a happy accommodation.

Are the TRegs in tumors the first kind, that are activated by the normal self-antigens that are present in the tumor cells (which are, remember, originally you to start with)? Or are they the second type, responding to the foreign antigen present in the tumor (mutated proteins, say, or over-expressed growth factors) but converted into a TReg type from a rejection-type when the tumor foreign antigens proved to be a chronic stuimulus?

A recent paper⁴ suggests it’s the latter:

This allows us to ask whether tumor-associated Treg cells arise from the repertoire of TCRs used by natural Treg cells or from the repertoire used by effector cells. We show that Treg population in tumors is dominated by T cells expressing the same TCRs as effector T cells. These data suggest that Treg in tumors are generated by expansion of a minor subset of Treg cells that shares TCRs with effector T cells or by conversion of effector CD4+ T cells and thus could represent adaptive Treg cells. ⁴

If this is generally true (and the authors do offer a helpful series of caveats) it has a very important implication. There’s a huge amount of interest in tumor vaccines — identify an antigen specific for the tumor, and induce a potent immune response to it, in the hope that T cells will then reject the tumor. But you see the problem: If the TRegs are stimulated by the same antigen, then your vaccine is going to increase both sides — the rejection branch and the TReg branch — and you’re no further ahead than when you started! This may be one of the reasons that tumor vaccines have been only intermittently effective. But it does make even more attractive another approach toward cancer immunization, where TRegs are specifically blocked, hopefully allowing the already-present rejection-type⁵ T cells to kick in and, maybe, eliminate the tumor:

This further suggests that improved cancer immunotherapy may depend on the ability to block tumor-antigen induced expansion of a minor Treg subset or generation of adaptive Treg cells, rather than solely on increasing the immunogenicity of vaccines. ⁴

1. I’m not quite comfortable with the phrasing here, but I can’t come up with a non-lawyerly, succinct way to phrase it. TRegs are part of the immune system, and so when they’re active the immune system isn’t blocked, it’s highly functional. What’s being blocked is what we traditionally think of as an immune response — the aggressive response that causes inflammation and that kills targets — while the TReg form is the branch of the immune response that prevents all those things. When TRegs are dominant, the immune response isn’t easily visible, but it’s still an active immune response.
2. Again, not happy with the term; if anyone has a more felicitious phrase, let me know
3. My qualifier here is “For now”, because this is a rapidly-changing field that has kind of outstripped my ability to follow it right now; I’m not quite sure whether this is the consensus view any more
4. Kuczma, M., Kopij, M., Pawlikowska, I., Wang, C., Rempala, G., & Kraj, P. (2010). Intratumoral Convergence of the TCR Repertoires of Effector and Foxp3+ CD4+ T cells PLoS ONE, 5 (10) DOI: 10.1371/journal.pone.0013623
5. Having typed that a dozen times here, I like it less than ever

There are two aspects about virology that constantly amaze me: How much we know about viruses, and how little we know about viruses.

Adenovirus research offers examples of both. Adenoviruses are probably among the best-studied virus groups.¹ We really do know an amazing amount about them. But it was only last year that Linda Gooding’s group offered the most convincing demonstration yet that adenoviruses actually establish a truly latent infection — a really basic aspect of their lifestyle, ² and a new paper from her group³ is looking at some equally-basic implications of that finding. (I talked about Gooding’s earlier latency finding here.)

It’s been known pretty much since day 1 that adenoviruses persistently infect tonsils;⁴ that was why they were first isolated, when the virus grew out of apparently-normal tonsil tissue in culture. The critical distinction is between mere “persistence” and true “latency”. In a latent infection, the virus shuts down production of new viruses, and is maintained basically as DNA within the host cell. Persistence is cruder — the virus continues to replicate, but at a low level that balances its destruction. Simplistically, latency is a destruction-free process, while persistence can include viral and cellular destruction.

Adenoviruses establish their latency in tonsils, which of course have lots of lymphocytes, but we usually think of adenoviruses as infecting epithelial-type cells, or hepatocytes, or whatever. Clinically, these guys typically cause cold-type symptoms, which you tend to get from fairly superficial infections of the respiratory tract lining. We don’t tend to think of adenoviruses as effective infectors of lymphocytes, but it turned out that their latent infection was, in fact, in T lymphocytes.  It looks like adenoviruses have one cell type (epithelial-type cells) for a lytic infection that leads to shedding of infectious virus, and another cell type for latent infection, allowing the virus to remain in the host and potentially re-infect an epithelial type later on.

Accordingly, Gooding and her team set up infections of cultured T lymphocytes in vitro, to see what would happen. In particular, they wanted to know whether, and how, the viral replication cycle would be controlled; and whether and how the host cell would be affected by the infection. I will skip over most of their findings and and highlight a couple that surprised me:

(1) The “Occupied!” sign. To get into a cell, adenoviruses usually need to bind to their cellular receptor, the CAR receptor.⁵ But latently-infected cells almost permanently shut off this receptor. For hundreds of days after the initial infection, cells express little or no CAR. The latent virus doesn’t want any competition; it has found a congenial long-term environment, and it doesn’t want some interloper infecting its cozy cell and perhaps destroying it.

There seem to be several mechanisms for the shutdown, but at least part of it is that the virus apparently permanently modifies the host DNA:

CAR synthesis and expression remained repressed even after the viral genome was lost (Fig. 8 and data not shown), suggesting a virus-induced epigenetic change to the cells that does not require the continued presence of the virus.³

And in fact the CAR isn’t the only thing to be modified for this purpose:

Even when CAR levels were restored by transduction with a CAR-containing retrovirus, the previously infected cells could not be reinfected³

We don’t know how the latent viruses were blocking superinfection, but it’s clear that the latent viruses really don’t want company.

(2) Rearranging the furniture.  The latent virus doesn’t stop at hanging an “occupied” sign; it modifies its host cell in other ways as well, apparently again by long-term or even permanent epigenetic modification of the DNA. That means that even after the virus itself is altogether gone, not even latently present, there are modified cells hanging about:

Remembering that adenoviruses infect just about everyone, that may mean that we’re all walking around carrying cells that are tagged and functionally altered by these viruses.

There’s been speculation for many years that adenovirus infection may underlie some forms of human tumors. One argument against this has been that there’s no evidence of adenovirus DNA in tumors, for the most part.⁶ (One rule of thumb in determining if a virus is actually causing a tumor is if it’s actually present in the tumor.) But of course, if adenoviruses leave a permanent scar on cellular DNA that lasts longer than the virus itself, this may not be relevant:

One compelling reason to gain an understanding of this nonlytic infection is the likelihood that adenovirus gene products cause damage to the host cell genome. … While these functions are irrelevant to the lytic infection of epithelial cells where all infected cells die, they are of serious concern when infected lymphocytes have carried the viral genome and survived. … Despite this normal appearance, the cells display altered gene expression long after the virus is lost.³

1. There are over 40,000 papers on adenoviruses, or at least mentioning them, in PubMed.
2. To be fair, it’s been suspected for decades that they do go latent, but that was the first time it was actually proven.
3. Zhang, Y., Huang, W., Ornelles, D., & Gooding, L. (2010). Modeling Adenovirus Latency in Human Lymphocyte Cell Lines Journal of Virology, 84 (17), 8799-8810 DOI: 10.1128/JVI.00562-10
4. I’m going to limit this discussion to the Group C adenoviruses — the latency concept may be true for other groups of adenoviruses but that hasn’t been directly shown.
5. “CAR” stands for the “Coxsackie B virus and Adenovirus Receptor”. Can anyone guess what other virus uses this receptor? Bueller? Anyone?
6. Also, the epidemiological links between tumors and adenoviruses are not very strong, at least in humans.

How does the immune response know when it’s needed? It has to eliminate problems, which means it needs to detect problems. So, what’s a problem?

In general, the immune system perceives two conditions as “problems”. One is when microbes are detected, and another is when damage is detected. These conditions are both detected through specific sets of receptors, and both lead to similar cascades of events that culminate in the response we think of as classically “immune” – first an innate immune response, and then if appropriate, an adaptive immune response that’s triggered by the initial innate response.

I’ve talked before about these two forms of problem detection. To summarize and grossly oversimplify some of the history: Charlie Janeway predicted the first form, which we now call “Pathogen-associated molecular pattern” (PAMP) detection; Polly Matzinger predicted the latter form, which we can call “Danger-associated molecular pattern” (DAMP) detection. PAMPs include things that are unique to bacteria or viruses — cell wall components that are present in bacteria, but not in vertebrate cells, for example; or double-stranded RNA, which is found in lots of viruses but would be unusual in our own cells. “Danger” signals, on the other hand, are indications of cell death — internal components of a cell, for example, that have leaked out as the cell dies. ¹ For a while, it looked as if PAMPs were the major signal leading to innate and then adaptive immunity, but more recently it’s become clear that DAMPs are also very important.

One example of DAMP recognition would be tumor recognition. We know that tumors are recognized by the immune system — by T cells and B cells, which are adaptive immunity. We know that adaptive immunity is very inefficient without an innate response to set up the proper conditions. We also know that tumors aren’t pathogens as such, and so you wouldn’t expect them to trigger PAMP receptors. So what’s triggering the immune response to the tumor? The answer seems to be DAMPs. As tumor cells die, which they tend to do much more exuberantly than normal cells, they release internal components that the immune response registers as evidence of danger. ² It’s even been proposed that the massive tumor cell death caused by chemotherapy is the real reason chemo works: The cell death is detected by the immune system as evidence of massive danger, and it’s the resulting immune response that actually eliminates the tumor, not the chemo per se.

Neutrophil

So, historically, DAMPs are DAMPs and PAMPs are PAMPs, and never the twain shall meet. After all, internal cell components are quite different from microbes, right?

Well, except for the internal cell components that actually are microbes. Mitochondria, of course, are actually exceedingly symbiotic bacteria that live inside our cells, right? And it turns out that, yes, some DAMPs actually are PAMPs, because some of the danger responses are actually triggered by mitochondrial components that are really bacterial in origin. A lovely paper from Carl Hauser’s lab³ shows that mitochondrial components, released from cells after damage, trigger innate immune responses through pathways that are more traditionally associated with pathogen-specific patterns.⁴

As I say, immune responses can be very destructive, and Hauser’s interest in this arises from the destructive aspect.  Trauma that produces lots of tissue damage can lead to severe inflammation that looks a lot like sepsis, even though there are no bacteria involved, so he has been looking for triggers for this sterile systemic inflammatory response syndrome (SIRS):

Cellular disruption by trauma releases mitochondrial DAMPs with evolutionarily conserved similarities to bacterial PAMPs into the circulation. These signal through innate immune pathways identical to those activated in sepsis to create a sepsis-like state. The release of such mitochondrial ‘enemies within’ by cellular injury is a key link between trauma, inflammation and SIRS.³

I found it particularly interesting  that one of the mitochondrial DAMPs is formylated peptides. Formylation of peptides is typical of bacteria, not eukaryotes, so it’s a good way of detecting pathogens. Indeed, there are receptors for formyl peptides on neutrophils (FPR1), among other cells, and the mitochondrial DAMPs (including the formyl peptides) cause neutrophils to migrate toward the source (chemotaxis) — see the movie below:

(Compare to this other movie I posted a while ago, which shows neutrophils in a mouse’s ear, being attracted to areas of tissue damage.)

H-2M3 crystal structure5

In fact, formylated peptides have been long known to be a PAMP, but not just via the FPR1; they’re also presented by a mouse non-classical MHC class I molecule, H-2M3.  (I didn’t include a picture of H-2M3 in my Field Guide to the MHC earlier, so here’s a picture to the left. ⁵ Heavy chain in grey, beta₂-microglobulin⁶ in red, peptide in green with the formylated end of the peptide — see how neatly it tucks into the peptide-binding groove there? in magenta.) And again, H-2M3 presents formylated peptides, not just from bacterial pathogens, but also from mitochondria. ⁷

Most people probably don’t think of MHC-family molecules as innate immune detectors, but many of the non-classical MHC molecules are true PAMP receptors (pattern recognition receptors, PRRs). It’s even been argued — based on H-2M3 itself, in fact — that this broad pattern-recognition ability is the original function of MHC molecules, and the role of MHC molecules in adaptive immunity is the latecomer:

F. M. Burnet asserted that it was their polymorphism that made MHC genes biologically significant. Certainly this is true for I-a⁸ function, but modern PRR-like I-b molecules⁹ suggest an alternate model for MHC origins. … Because most genes are monomorphic or minimally oligomorphic, and most class I-like genes not linked to the MHC are monomorphic, parsimony suggests the ancestral MHC locus was also monomorphic. This primitive MHC molecule, functioning as a PRR, would have been preadapted for the evolution of polymorphic class I-a molecules in the evolving adaptive immune system. ¹⁰

1. In apoptosis, the programmed cell death that’s a normal part of tissue growth, internal cell components are carefully packaged up in such as way as to prevent this kind of response.
2. Which, of course, it is.
3. Zhang, Q., Raoof, M., Chen, Y., Sumi, Y., Sursal, T., Junger, W., Brohi, K., Itagaki, K., & Hauser, C. (2010). Circulating mitochondrial DAMPs cause inflammatory responses to injury Nature, 464 (7285), 104-107 DOI: 10.1038/nature08780
4. Thanks to Alex Ling, who wrote to me suggesting I talk about this paper.  I’d filed it in with the other 512 papers that I want to talk about here, some time, but her email made me take another look and appreciate how neat the work is.
5. Wang, C. R., Castano, A. R., Peterson, P. A., Slaughter, C., Lindahl, K. F., and Deisenhofer, J. (1995). Nonclassical binding of formylated peptide in crystal structure of the MHC class Ib molecule H2-M3. Cell 82, 655-664.
6. Why doesn’t the beta symbol ? stick? No matter how often, or how, I enter the code, it changes to a ? in the published post.
7. Loveland, B., Wang, C. R., Yonekawa, H., Hermel, E., and Lindahl, K. F. (1990). Maternally transmitted histocompatibility antigen of mice: a hydrophobic peptide of a mitochondrially encoded protein. Cell 60, 971-980.
   Shawar, S. M., Vyas, J. M., Rodgers, J. R., Cook, R. G., and Rich, R. R. (1991). Specialized functions of major histocompatibility complex class I molecules. II. Hmt binds N-formylated peptides of mitochondrial and prokaryotic origin. J. Exp. Med. 174, 941-944.
8. I-a are the classical MHC class I molecules that present peptides to cytotoxic T lymphocytes
9. And, logically enough then, I-b are the non-classical MHC molecules that often present fairly conserved molecules.
10. Doyle, C. K., Davis, B. K., Cook, R. G., Rich, R. R., and Rodgers, J. R. (2003). Hyperconservation of the N-formyl peptide binding site of M3: evidence that M3 is an old eutherian molecule with conserved recognition of a pathogen-associated molecular pattern. J. Immunol. 171, 836-844.

What’s a tumor?

In some ways, that’s a bad question (never mind the answer) because it implies that a tumor is a single thing. But we know that’s not true. A tumor, by the time we can detect it, is a collection of many cells, at least billions of them, and those cells are not all the same. I’m not even talking about the normal cell types that are incorporated into a tumor (things like blood vessels and support cells). Even cells that are unambiguously cancerous are very different within a tumor. And of course, that’s important for the things we’re most interested in, prognosis and treatment, because it’s not the average tumor cell that we’re most concerned about, it’s the subset of tumor cells that are most resistant to treatment, or that are most aggressive.

The development of this variation is really fundamental to how we understand tumor formation and tumor growth. Cancerous cells don’t just appear, fully ready to metastasize and grow. What happens is that a normal cell mutates slightly and gains a little advantage. Most of its progeny stay like that, but one of them mutates again and changes a little more, and then one of that cell’s progeny mutates again, and so on. It probably takes at least a half-dozen mutations, over many cell generations, before a normal cell has progressed through to a detectably cancerous cell.  (I’ve talked about this before, here.)

Also, since truly normal cells simply don’t mutate that many times — there are too many checks and repair systems to allow a half-dozen mutations to accumulate in a single human’s² lifetime — one of the mutations is probably in the check/repair system, turning the cancerous pathway into a mutator pathway as well.

So we expect tumors to be made up of many different cell types, and this is indeed what we see:

With rare exceptions, human malignancies are thought to originate from a single cell, yet by the time of diagnosis, most tumors display startling heterogeneity in cell morphology, proliferation rates, angiogenic and metastatic potential, and expression of cell surface molecules. ¹

So how diverse are tumors?

That’s been a hard question to answer, because you’d need tools to look at individual cells, and you’d also need some way of expressing that diversity. A recent paper¹ looked at diversity in breast cancer using some individual-cell tools, which I’m not going to discuss, and took an interesting approach to describing the variability:

… we applied diversity measures from the ecology and evolution sciences to our copy number data. These diversity measures estimate the number and distribution of species in a certain geographical area or environmental niche. In our context, a species is a cancer cell population … Hence, a region of a tumor containing cancer cells with 3 different copy number ratios is interpreted to contain 3 distinct “species.” ¹

They suggest that this way of describing tumors could be a useful aid to prognosis and to predicting response to therapy, offering a quantitative description of tumor variability (which might correlate with the tumor’s potential for spread and escaping treatment).

I hadn’t thought of tumors as ecosystems before, but I wonder if the analogy could be taken further by considering, say,cytotoxic T lymphocytes as predators …

1. Park, S., Gönen, M., Kim, H., Michor, F., & Polyak, K. (2010). Cellular and genetic diversity in the progression of in situ human breast carcinomas to an invasive phenotype Journal of Clinical Investigation DOI: 10.1172/JCI40724
2. let alone a mouse’s lifetime

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.

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

Tumors are supposed to be destroyed by our immune system. So how come we still see tumors?

A big part of the answer is probably that our immune system is very good at destroying proto-tumors, but is not so good at handling those that manage to sneak through and grow to the point of detectability. That splits the first question into two questions: Why do some proto-tumors manage to sneak through, not being eliminated by the immune system? And why is it that detectable tumors are not effectively handled?

The first part, I think, may often be related to cell-intrinsic immune escape mutations. That is, pre-cancerous cells are constantly being attacked by the immune system; in turn (if they survive long enough) they constantly mutate, doing things like damaging the antigen-presentation pathway that makes them recognizable by the immune system. Eventually, they find some configuration that reduces the rate at which they’re killed. Once cancer cell replication is even fractionally greater than destruction,¹ a tumor can begin to grow.

So that’s probably the earliest stage of tumor growth. But once tumors reach a certain size, a second factor kicks in. Chronic immune responses are dangerous; after all, the whole point of the immune system is to kill things. The chronic immune response against the growing tumor is now shut down. This has been understood for quite a while — the immune system often becomes “tolerant” of a tumor. More recently, it’s become clear that it’s not merely “tolerance” (which implies that the immune system is simply benignly ignoring the tumor); the presence of a tumor actively forces the immune system to shut itself down, slamming on the brakes rather than just peacefully coasting by.

Brakes are a fundamental part of an active immune response. If you look at diagrams of normal immune responses, they show inverted “U” shaped curves (in here and here, for example), where the response is triggered, rapidly ramps up, hopefully does its thing, and then just as rapidly shuts down to near-background levels once again. There used to be a sort of general feeling that this was a rather passive thing — pathogen stimulates response, response destroys pathogen, no more stimulus, response goes away — but now we understand that the shut-down phase is just as active and dynamic as the upward curve. Just as with the upward phase, there are all kinds of different mechanisms to control the response; one of the most important is the “Regulatory T cell” (TReg).  And it’s pretty clear that TRegs are involved in controlling the immune response to tumors (I talked about that here, and links therein).

TRegs have been known for a while (I gave a brief history, including the I-J fiasco, here). The usual description of a TReg includes a number of markers;² one of the most basic is CD4. CD4 T cells used to be lumped together as “T Helper” cells, but now we have multiple sub-specialties in the CD4 category, and TRegs are one of those specialities.

More recently, TRegs — or at least cells that function the same way as TRegs — have been described in the CD8 population of T cells.³ CD8 T cells are traditionally called “Cytotoxic T lymphocytes” (CTL) (although it’s been increasingly clear that cytotoxicity is just one of many functions a CD8 T cell can offer), but it seems that these variants of CD8s can actively shut down an ongoing immune response, in a specific and targeted way. There seems to be a trend to calling these cells “suppressor cells” rather than “TRegs”. “Suppressor T cells” is an older term that was out of favor for a while, but it’s probably useful to bring it back and distinguish between the natural TRegs and some of the other cells that can do something similar but that have different sources and origins.

At least some of the CD8 suppressor T cells can arise from apparently-conventional CD8 T cells. That is, you can pull CD8 T cells out of a normal mouse’s spleen, and depending on what those cells see and are exposed to, they could progress to being conventional CTL — killing tumor cells, producing interferon and other cytokines, generally being a destructive force — or they could become suppressor CD8 T cells, and actively prevent that destruction from happening.

It turns out that one of the forces that can drive a CD8 T cell into being a suppressor T cell is a tumor. A recent paper from Arthur Hurwitz’s lab⁴ shows this quite clearly. They had shown previously that transferring specific CD8 T cells into a tumor-bearing mouse resulted in what they called “tolerance”.⁵ But now they demonstrate that it’s more than that; the transferred CD8s are converted into suppressor T cells that actively shut down immune responses.

Tumor-infiltrating TcR-I cells suppressed the in vitro proliferation of both melanoma Ag-specific CD8+ (37B7) T cells and OVA-specific CD4+ (OT-II) T cells. … Even at a ratio of one TcR-I cell to four responder T cells, we observed 30% suppression of proliferation. ⁴

This isn’t the only way that tumors escape immune recognition, but (at least for some tumors) it may be an important one. It’s clearly an important consideration for things like tumor vaccines and immune therapy, because it suggests that immunizing with tumor antigens (and thereby generating lots of tumor-specific CD8 T cells) may actually increase the suppressive effect of the tumor.

The conversion of CD8+ effector T cells into suppressor cells may be one mechanism by which tumors restrict the immune response from effectively controlling tumor growth. As subsequent effectors infiltrate the tumor, either following peripheral sensitization or as a result of adoptive transfer therapy, the induced regulatory cells may suppress these new effectors and reduce their ability to confer tumor immunity. This cyclic suppressive process may contribute to the profound loss of antitumor responses following adoptive immunotherapy. ⁴

(My emphasis.) On the other hand, if this is a common mechanism, then overriding it — which should be possible, using cytokines, specific T cell subsets, and/or targeted receptor ligands — may switch the suppressive population abruptly back into an effector group, turning the brainwashed traitors into resistance fighters.

1. Destruction would include far more than immune destruction, of course — it would include cells that become differentiated and no longer replicated, cells that outgrow their oxygen supply, cells that undergo apoptosis, and so on
2. FoxP3, CD25, and so on
3. I’m not sure who made the first identification; this looks as if it’s one of those fields where there were incremental advances, hinting more and more strongly at the presence of these cells, but with no single clearcut starting point. Papers in the early 2000s start to point at regulatory CD8s, and by 2004 a handful of relatively high-profile papers fairly solidly identified them. A 2004 review paper is
   Zimring, J., & Kapp, J. (2004). Identification and Characterization of CD8+ Suppressor T Cells Immunologic Research, 29 (1-3), 303-312 DOI: 10.1385/IR:29:1-3:303
4. Shafer-Weaver, K., Anderson, M., Stagliano, K., Malyguine, A., Greenberg, N., & Hurwitz, A. (2009). Cutting Edge: Tumor-Specific CD8+ T Cells Infiltrating Prostatic Tumors Are Induced to Become Suppressor Cells The Journal of Immunology, 183 (8), 4848-4852 DOI: 10.4049/jimmunol.0900848
5. Anderson MJ, Shafer-Weaver K, Greenberg NM, & Hurwitz AA (2007). Tolerization of tumor-specific T cells despite efficient initial priming in a primary murine model of prostate cancer. Journal of immunology (Baltimore, Md. : 1950), 178 (3), 1268-76 PMID: 17237372