The current understanding says “Yes”, but with reservations. As I’ve noted in previous posts (here and links therein, among others), there’s pretty solid evidence now that the immune system controls tumors in their early development.

Probably (we don’t know this for sure, but evidence points to it) there are many proto-tumors that begin to form, taking a few steps along the long route to full-blown cancer, but that are destroyed by the immune system long before they ever become detectable. Probably many more tumors form and take those early steps, and though they are not completely eliminated by the immune system they are controlled — the immune system prevents the proto-tumor from ever becoming more than a little cluster of cells, even though that little cluster of cells may persist for many years.

By the time a cancer is clinically detectable, though, does the immune system have any effect? Again referring to the current model, proto-tumors are able to advance to the detectable stage because they have avoided immune control. Therefore, the tumor we see are almost by definition uncontrollable by the immune system, right?

Immune control of clinical tumors?

Actually, that’s not quite what the theory suggests. A tumor that’s reached the detectable level grows faster than the immune system shuts it down, true; but that doesn’t mean there’s no influence of the immune system. Yes, the tumor could be growing twice as fast as it should, with no influence of the immune system. But equally, the tumor could be growing 10 times too fast, with the immune system destroying 90% of that. The overall rate would look the same; but in the latter case, we only need to push the growth rate down, or crank up the immune response, by 11%, to drive the tumor into remission.

Is there any direct evidence that the immune system slows the progression even of outright cancer? Certainly there is, though most of the evidence I know of it a little circumstantial. One line of reasoning is that, if the immune system controls tumors, then we should see a correlation between immune responses to tumors, and their prognosis. In fact, there are quite a few papers that show that: For example, a paper in Clinical Cancer Research last month.¹

This group actually looked at two parameters, that might or might not be connected, and their influence on prognosis of ovarian carcinoma. On the one hand, they looked at evidence for tumor immune evasion: How stringently was the tumor avoiding cytotoxic T lymphocyte recognition? On the other hand, they looked at infiltrating T cells in the tumor: How well could T cells recognize the tumor?

(To my mind, the latter is a much more important question, because we don’t know much about thresholds and cumulative effects of immune evasion — that is, we aren’t yet able to look at the recognition molecules as such, and declare that T cells will or will not recognize the tumor. Of course, this sort of study, that correlates phenotype and function, will be critical for answering that question.)

Immune evasion is bad for survival

Impressively, there’s a strong link between good prognosis and phenotype. Tumors that seems to have good antigen presentation, have a better prognosis than those that have apparently blocked their antigen presentation pathways efficiently. (They were able to break it down further than that, to the specific types of molecules that may be important.) And these are not trivial differences; people with defective antigen presentation survived for 1 or 2 years, those with good antigen presentation averaged 4 or 5 years or longer.

Patients with all five markers positive in the tumor lived almost four times as long (median survival 5.67 years; P < 0.01) and were 4.74 times less likely to die from their disease

The other half of the study was almost equally impressive.

Patients with complete absence of tumor-infiltrating T cells were 2.04 times more likely to die from their disease (95% CI, 1.35-3.07) than those with one or more T cells (P < 0.01; median survival 1.67 years versus 3.79 years). … [ However,] Although peritumoral presence of CD3+/CD8+ T cells was a significant survival factor in the univariate analyses limited to patients with advanced-stage cancer, it did not emerge as a significant factor in multivariate analyses.

This sort of study can’t definitively answer the question of whether there’s any significant control of clinically detectable cancers. For example, since there’s evidence that chemotherapy success is linked to the immune response, perhaps the immune parameters here are actually measuring the efficacy of chemotherapy, and the immune response is ineffective on its own. Still, it’s certainly encouraging — it suggests that the immune system really is a potential partner in treatment of many tumors, and maybe gives a pointer to which tumors are more or less likely to respond to treatment.

1. Han, L.Y., Fletcher, M.S., Urbauer, D.L., Mueller, P., Landen, C.N., Kamat, A.A., Lin, Y.G., Merritt, W.M., Spannuth, W.A., Deavers, M.T., De Geest, K., Gershenson, D.M., Lutgendorf, S.K., Ferrone, S., Sood, A.K. (2008). HLA Class I Antigen Processing Machinery Component Expression and Intratumoral T-Cell Infiltrate as Independent Prognostic Markers in Ovarian Carcinoma. Clinical Cancer Research, 14(11), 3372-3379. DOI: 10.1158/1078-0432.CCR-07-4433

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

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

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

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

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

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

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

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

But perhaps I should begin at the beginning.

Proteolysis is a normal part of cell function. Proteins that are damaged, misfolded, or mis-translated, as well as proteins that have simply reached the end of their useful life, are degraded and converted to amino acids that can be recycled into new proteins. In the early- to mid-1990s, there were three general systems that were known to degrade proteins, depending on which subcellular compartment they were in:

• In the cytosol and nucleus, proteins are predominately degraded by proteasomes.
• Proteins taken up from the exterior of the cell can be degraded in acidic lysosomes
• Mitochondrial proteins are degraded by a number of proteases within the mitochondrion¹

That leaves an obvious gap. What happens to proteins that are in the endoplasmic reticulum (ER)? This is particularly relevant because the ER is a ferociously active site of protein synthesis, folding, and assembly; when any of those steps goes awry, the protein is supposed to be degraded, a process known as “quality control”. It was clear in the 1980s that proteins that failed quality control in the ER were degraded; in human cells, a well-known example was the cystic fibrosis transmembrane conductance regulator (CFTR), which folds inefficiently and is rapidly degraded². But it was not clear where the degradation happened (in the ER? The cytosol? Somewhere else?), and what proteases were responsible.

At first it was believed that “what happens in the ER stays in the ER” — ER proteins were degraded in the ER, by ill-defined proteases in that compartment. But I don’t think there was much enthusiasm for that belief, and basically, the field was a mess, as you can see from this introductory paragraph from the time:³

Other membrane proteins are also known to be degraded at the ER, but the process is poorly understood, and the responsible enzymes have not been identified. For example, some of these proteolytic events are ATP dependent, but some are not; some occur within the lumen while others take place on the cytoplasmic side; some exhibit inhibitor sensitivities characteristic of serine proteases, whereas others do not.

This was relevant to me as an immunologist, because viral glycoproteins (which, of course, are synthesized in the ER) are popular targets for cytotoxic T lymphocyte (CTL) recognition (a quick review of MHC class I antigen presentation is here). We knew in the early 1990s that most if not all CTL targets — even those derived from ER proteins — were generated in the cytosol. As I wrote in a 1996 review:⁴

Proteins targeted into the ER by signal sequences can also be presented on MHC I molecules. Since these molecules are cotranslationally transported into the endoplasmic reticulum, they might be expected to bypass hydrolysis in the cytosol. However, where analyzed, the presentation of most of these antigens is dependent on the TAP-transporter and on proteasome activity, and therefore the presented peptides are probably being generated in the cytosol.

The three obvious possible explanations were that either the putative glycoprotein never made it into the ER and was degraded as a mistargeted protein (Jon Yewdell would call that a “DRiP”; I can’t remember exactly when I heard him propose that first, but it was around that time); that the glycoprotein went in to the ER, got degraded there, and the peptides were first transfered to the cytosol; or that the glycoprotein was transfered from the ER to the cytosol and degraded there.

The simplest explanation, at the time, seemed to be the first one –  proteins never made it in to the ER, and were degraded in the cytosol. However, it wasn’t an explanation that we liked very much, for various reasons. Vic Engelhard’s paper (Remember Engelhard’s paper? This here’s a post about Engelhard’s paper) cleared that question up, at least for one epitope.

Skipper, J. C., Hendrickson, R. C., Gulden, P. H., Brichard, V., Van Pel, A., Chen, Y., Shabanowitz, J., Wolfel, T., Slingluff, C. L., Jr., Boon, T., Hunt, D. F., and Engelhard, V. H. (1996). An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. J. Exp. Med. 183, 527-534.

His finding is simple enough to describe, although it relied on a technically very difficult mass spec analysis:

1. A peptide presented on MHC class I was derived from an ER protein (which they knew from its sequence);
2. the protein had actually gone into the ER, because it had been N-glycosylated, which only happens in the ER;
3. yet the peptide itself was probably generated in the cytosol, because enzymes that modified it are mainly found in the cytosol.

The most surprising and exciting part was the second point: Clear evidence that the protein had actually gone into the ER before the peptide was generated. ⁵ This wasn’t by any means definitive proof that ER proteins are degraded in the cytosol (a follow-up paper in 1998⁶ took it a bit further) but it certainly was suggestive.

If Vic’s paper had come out a year or two earlier, it would probably have made much more of a splash than it did, but in February of 1996 it was only a nose ahead of several more focused papers. The field had started to clear up in the mid-1990s, with some observations in yeast in 1993 and 1994 ⁷ and around 1995 moving into mammalian cells with (among others) the Jensen et al. paper I quoted above. ⁸  And later in 1996, the iceberg tipped over altogether, with a whole bunch of almost simultaneous papers that showed quite clearly that ER degradation wasn’t done by ER proteases at all, but was in fact performed by proteasomes. ⁹ All the papers demonstrated that there’s an export step before degradation: ER proteins that fail quality control are shunted out into the cytosol, where the proteasomes can grab onto them and chop them up. (The export step is still not all that well understood in molecular detail, though in 2007 it started to open up some, I think.)

Though Engelhard’s 1996 paper is reasonably widely cited (256 citations as I write this) it clearly didn’t have the impact on cell biology in general that it did on me, probably because it came out around the same time as a bunch of more specific papers. This blogpost is an attempt to give a bit more retroactive credit to a very nice example of logical reasoning from indirect evidence.

1. I believe that some, though not all, of the mitochondrial proteases were identified in the late 1980s/early 1990s, and that the broad outline of mitochondrial proteolysis was understood in the early 1980s (Desautels, M. and Goldberg, A. L. (1982) Liver mitochondria contain an ATP-dependent, vanadate-sensitive pathway for the degradation of proteins. Proc Natl Acad Sci USA 79 , pp. 1869-1873.). I don’t know all that much about mitochondrial proteolysis, though, so if someone wants to correct me, please do so.
2. For example, Ward C, Kopito R (October 14, 1994) Intracellular turnover of cystic fibrosis transmembrane conductance regulator. Inefficient processing and rapid degradation of wild-type and mutant proteins. J. Biol. Chem. 269.:25710-25718
3. Taken from Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, et al. (October 6, 1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83:129-35. with references removed
4. York, I. A., and Rock, K. L. (1996). Antigen processing and presentation by the class I major histocompatibility complex. Annual review of immunology Annu Rev Immunol 14, 369-396.
5. Technical explanation: The mass spec analysis showed that the asparagine that is encoded in the DNA was actually an aspartic acid in the presented peptide; deglycosylating enzymes that were believed to only be present in the cytosol remove carbohydrates from Asn to generate Asp.
6. Mosse, C. A., Meadows, L., Luckey, C. J., Kittlesen, D. J., Huczko, E. L., Slingluff, C. L., Shabanowitz, J., Hunt, D. F., and Engelhard, V. H. (1998). The class I antigen-processing pathway for the membrane protein tyrosinase involves translation in the endoplasmic reticulum and processing in the cytosol. J Exp Med 187, 37-48.
7. (Sommer T, Jentsch S (September 9, 1993) A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum. Nature 365.:176-9.
   and
   Kölling R, Hollenberg CP (July 15, 1994) The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants. EMBO J 13.:3261-71.
8. Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, et al. (October 6, 1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83:129-35.
9. I may be missing some:
   Hampton RY, Gardner RG, Rine J (December 1996) Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. Mol Biol Cell 7.:2029-44.
   Werner ED, Brodsky JL, McCracken AA (November 26, 1996) Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate. Proc Natl Acad Sci U S A 93.:13797-801.
   Hiller MM, Finger A, Schweiger M, Wolf DH (September 20, 1996) ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway. Science 273.:1725-8.
   Qu D, Teckman JH, Omura S, Perlmutter DH (September 13, 1996) Degradation of a mutant secretory protein, alpha1-antitrypsin Z, in the endoplasmic reticulum requires proteasome activity. J Biol Chem 271.:22791-5.
   Wiertz EJ, Jones TR, Sun L, Bogyo M, Geuze HJ, et al. (March 8, 1996) The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell 84.:769-79.

What has been surprising is how ineffective these viral immune evasion mechanisms seem to be. (I’ve talked about this before in a bit more detail.) In cells in the incubator (which is where most of the experiments with the antigen presentation immune evasion molecules have been done), the effects often look pretty dramatic; but when you look at the immune response in humans infected with human cytomegalovirus¹ or herpes simplex virus² (both of which have formidable immune evasion functions in tissue culture³) there are pretty hearty and effective CTL responses, suggesting that the immune evasion doesn’t work all that well in vivo.

Most of the viruses that have had immune evasion genes identified are human viruses; that’s where the clinical interest and the money are. One problem with this is that the large DNA viruses that are likely to have these immune evasion functions tend to be pretty species-specific. Human herpesviruses and adenoviruses don’t infect mice well, if at all, and even if they do infect lab animals it’s not really clear how accurately this infection mimics the natural course in humans. There are a couple of lab animal herpesviruses that may be useful models (though even here the effects of immune evasion molecules are quite small.⁴) Still, it would be nice to understand what’s going on with immune evasion in, say, herpes simplex infection.

Herpes simplex virus actually will infect mice (the alphaherpesviruses tend to be somewhat less host-restricted than the beta or gammaherpesviruses), and while mouse infection isn’t exactly like that in humans it does seem to be similar enough to offer useful insights. Not into immune evasion, though, because the immune evasion gene of herpes simplex (called “ICP47″) works very poorly in mice.⁵ Recently, though, Chris Wilson’s group took an ingenious approach to looking at the question, and a theme may be starting to emerge.

Wilson reasoned that if ICP47 doesn’t work in mice,⁶ why not replace it with a functionally-similar gene that does? They made recombinant herpes simplex viruses that have immune evasion genes from mouse or human cytomegalovirus that block mouse MHC class I fairly well. These viruses are more neurovirulent in mice than the control viruses⁷ and this was because the CTL didn’t work as well in the recombinants. ^8,9

Their latest paper¹⁰ shows something new and exciting with the recombinant virus: It reactivates better from neurons.

Trigeminal ganglion

Remember that HSV establishes a latent infection in neurons and, in humans, periodically reactivates and may shed and infect new hosts this way. HSV latency is still a mysterious and little-understood field in spite of huge amounts of work over the years, but a host of recent findings have started to explain at least bits and pieces. It used to be felt that latent HSV didn’t express any proteins, and so should be invisible to the immune system. As I observed here, it’s now known that the immune system can detect HSV in neurons. That offers a mechanism for reactivation to be coupled with immune suppression. The Orr et al paper here offers more support for this concept, and suggests that one of the (most important?) functions for HSV immune evasion is to tip the scales a little further toward the virus being able to escape recognition in the neuron, and therefore being more easily able to reactivate and infect new hosts — something this ubiquitous virus does with extraordinary efficiency. Orr et al call this an “actively testing the waters” model, in which HSV constantly sticks its toes out into the stream of the immune response to see if it’s safe to reactivate.

What especially interesting about this is that we now have (that I know of) three models of MHC class I immune evasion in more or less authentic infections, and each case the immune evasion seems to be designed to support latency, reactivation, and transmission. Here we have HSV using immune evasion in latency and persistence. Ann Hill’s group has recently shown that mouse cytomegalovirus MHC class I immune evasion proteins help the virus persist in the salivary glands,¹¹ so that (as with the HSV story) it is more able to infect new hosts. And in the third model I know of, mouse herpesvirus 68, the MHC class I immune evasion genes are important for, you guessed it, latency and persistence.¹²

Three data points aren’t very many, but it does suggest that this may be a common feature of the herpesviruses. Perhaps in acute infection the virus doesn’t really “want” to block immune responses all the effectively, because their lifestyle involves only a brief burst of acute infection followed by a long-term latency; and it’s that latency that allows infection of new hosts. The viruses “let” themselves be driven into apparent submission, but then use their immune evasion functions to mount intermittent stealth campaigns to recruit new hosts.

If this is true for herpesviruses (and of course it’s just a hypothesis) is it also true for other viruses that have MHC class I immune evasion functions? Maybe for some (I wonder about the adenoviruses in particular, since they’re also very capable of long-term persistence in spite of immune responses) but I doubt it’s universal — HIV, for example, doesn’t obviously fit into the same lifestyle pattern as the herpesviruses, and yet if has a protein (nef) that targets MHC class I. Still, if offers a framework for thinking about the problem, which is very valuable.

1. Manley, T. J., Luy, L., Jones, T., Boeckh, M., Mutimer, H., and Riddell, S. R. (2004). Immune evasion proteins of human cytomegalovirus do not prevent a diverse CD8+ cytotoxic T-cell response in natural infection. Blood 104, 1075-1082.
2. Posavad, C. M., Koelle, D. M., and Corey, L. (1996). High frequency of CD8+ cytotoxic T-lymphocyte precursors specific for herpes simplex viruses in persons with genital herpes. J. Virol. 70, 8165-8168.
3. York, I. A., Roop, C., Andrews, D. W., Riddell, S. R., Graham, F. L., and Johnson, D. C. (1994). A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell 77, 525-535.
   and
   Jones, T. R., Hanson, L. K., Sun, L., Slater, J. S., Stenberg, R. M., and Campbell, A. E. (1995). Multiple independent loci within the human cytomegalovirus unique short region down-regulate expression of major histocompatibility complex class I heavy chains. J. Virol. 69, 4830-4841.
4. For example, Munks, M. W., Pinto, A. K., Doom, C. M., and Hill, A. B. (2007). Viral interference with antigen presentation does not alter acute or chronic CD8 T cell immunodominance in murine cytomegalovirus infection. J Immunol 178, 7235-7241.
5. Jugovic, P., Hill, A. M., Tomazin, R., Ploegh, H., and Johnson, D. C. (1998). Inhibition of major histocompatibility complex class I antigen presentation in pig and primate cells by herpes simplex virus type 1 and 2 ICP47. Journal of Virology 72, 5076-5084.
6. To be strictly accurate, ICP47 does work in mice, just not very well — 100 times less than in humans, maybe, though what the unit of comparison is or should be isn’t clear
7. I first wrote “than wild-type virus”, but I don’t think we know that — the process of recombination made the virus less virulent and the comparisons were done to this reduced-virulence control virus; I don’t see a direct comparison to wild-type virus.
8. Orr, M. T., Edelmann, K. H., Vieira, J., Corey, L., Raulet, D. H., and Wilson, C. B. (2005). Inhibition of MHC class I is a virulence factor in herpes simplex virus infection of mice. PLoS Pathog 1, e7.
9. This actually parallels and supports previous work showing that ICP47 does in fact have a small effect on neurovirulence in mice.
10. ORR, M., MATHIS, M., LAGUNOFF, M., SACKS, J., WILSON, C. (2007). CD8 T Cell Control of HSV Reactivation from Latency Is Abrogated by Viral Inhibition of MHC Class I. Cell Host & Microbe, 2(3), 172-180. DOI: 10.1016/j.chom.2007.06.013
11. Lu, X., Pinto, A. K., Kelly, A. M., Cho, K. S., and Hill, A. B. (2006). Murine cytomegalovirus interference with antigen presentation contributes to the inability of CD8 T cells to control virus in the salivary gland. J Virol 80, 4200-4202.
12. Bennett, N. J., May, J. S., and Stevenson, P. G. (2005). Gamma-herpesvirus latency requires T cell evasion during episome maintenance. PLoS Biol 3, e120.