A while ago I listed a number of reasons why smallpox was eradicated, whereas other diseases haven’t been (yet). One of the reasons was that the vaccine against smallpox¹ is so effective. Vaccinia immunization induces immunity for an extraordinarily long time — memory immune responses have been shown for up to 60 years after vaccination.

So why is vaccinia such an effective vaccine? Part of it is that vaccinia is a live virus: It replicates after you’re inoculated (so there’s lots of antigen there), and it stimulates the innate immune response (which is geared toward detection of live viruses, among other things). (The yellow fever vaccine is another live virus vaccine that’s also famous for inducing long-term immunity.) Vaccinia is also a large virus that has a lot of antigens available, so that there are lots of different modes of immunity triggered. That is, both B cell (antibody-based) immunity, and broad T cell-based immunity, are likely to be present and to have lots of different targets.

A recent paper² suggests that the route of vaccination is also important. Unlike most vaccines, which are given by intramuscular (e.g. influenza vaccine) or subcutaneous (e.g. yellow fever) injection, or orally (the live polio vaccine), vaccinia is delivered by scarification — scraping the most superficial layers of the skin. I don’t think this was the result of deliberate comparisons — scarification was the traditional method, and it was easy and convenient. Before 1967:

A scratch about 5 mm long was made in the skin with a needle, a lancet or a small knife and the vaccine suspension was rubbed into the site. A single cut or cross cuts were made, in 1 , 2 or 4 different sites. This was essentially the same method as had been used for variolation in Europe during the latter part of the 18th century. ((Smallpox and its Eradication (Chapter 7). F. Fenner, D. A. Henderson, I. Arita, Z. JeZek, I. D. Ladnyi. World Health Organization, Geneva, 1988))

Later, a bifurcated needle was used:

Experiments soon showed that the multiple puncture method, in which the bifurcated needle was held at right angles to the skin, which was then punctured several times with the prongs, was very efficient and very easy even for an illiterate vaccinator to learn. It became the standard method of vaccination throughout the world. ³

Scarification was a simple and convenient way to deliver the vaccine.  It turns out that scarification isn’t just a convenience, it’s the most effective way to get immunity:

VACV immunization via s.s. [skin scarification], but not conventional injection routes, is essential for the generation of superior T cell–mediated immune responses that provide complete protection against subsequent challenges.²

Langerhans cells in the skin4

This includes protection against respiratory-spread disease, not just skin infection. My first thought was that this is probably simply because the vaccinia virus replicates better in the skin than by intramuscular injection, but the improved immunogenicity is also seen with a non-replicating version of vaccinia, “MVA”. ⁵

My next thought is that Langerhans cells are probably part of the reason. Langerhans cells (see the figure to the right) are a subset of dendritic cells, probably extremely good at triggering immunity, that form a dense network under the skin, and probably act very efficiently at filtering skin-delivered antigen and delivering it to the immune system.

Also, the fact that the skin is damaged in the process evokes Polly Matzinger’s “danger” concept of immune stimulation.

At any rate, something, even if we don’t know exactly what, about scarification leads to better immunity, at least for vaccinia virus. That’s useful to know. Having said that, I’m not quite sure why this paper appeared in Nature Medicine, a very high-impact journal — the mechanism wasn’t shown at all clearly, and this isn’t the first time that the general observation has been made:

This study strongly indicated that, although less reactogenic, vaccinia vaccine administered im [intramuscularly] at a dose of 10⁵ pfu fails to induce an immune response comparable to that elicited by standard scarification. ⁶

Even more broadly, the skin inoculation concept has been shown to lead to high immunogenicity in other systems; for example, it was shown a couple of years ago that yellow fever vaccine is more immunogenic when delivered intradermally than when given by its conventional subcutaneous route:

Intradermal administration of one fifth of the amount of yellow fever vaccine administered subcutaneously results in protective seroimmunity in all volunteers. ⁷

Bifurcated needle used for smallpox vaccination

(I do have to add that apparently scarification — which is much easier than intradermal injection — does not work for yellow fever, based on some experiments in the 1950s.⁸ I haven’t read those papers myself, though. I’d be interested to see if the bifurcated needles used in the late 1960s and on for vaccinia might be more effective for the yellow fever vaccine.)

Anyway, seeing this in at least two instances⁹ makes it seem possible that it’s a general effect. If skin administration enhances immunogenicity, perhaps this is a way of extending limited vaccine stocks in an emergency.

1. That is, vaccinia virus
2. Liu, L., Zhong, Q., Tian, T., Dubin, K., Athale, S., & Kupper, T. (2010). Epidermal injury and infection during poxvirus immunization is crucial for the generation of highly protective T cell–mediated immunity Nature Medicine, 16 (2), 224-227 DOI: 10.1038/nm.2078
3. Smallpox and its Eradication (Chapter 11).  F. Fenner, D. A. Henderson, I. Arita, Z. JeZek, I. D. Ladnyi.  World Health Organization, Geneva, 1988
4. MAHNKE, K., JOHNSON, T., RING, S., & ENK, A. (2007). Tolerogenic dendritic cells and regulatory T cells: A two-way relationship Journal of Dermatological Science, 46 (3), 159-167 DOI: 10.1016/j.jdermsci.2007.03.002
5. At any rate, it’s claimed to be non-replicating, but I don’t remember seeing it formally shown that MVA doesn’t replicate, even temporarily, in the skin. Anyone know if this has been tested?
6. Immunologic Responses to Vaccinia Vaccines Administered by Different Parenteral Routes Author(s): David J. McClain, Shannon Harrison, Curtis L. Yeager, John Cruz, Francis A. Ennis, Paul Gibbs, Michael S. Wright, Peter L. Summers, James D. Arthur, Jess A. Graham Source: The Journal of Infectious Diseases, Vol. 175, No. 4 (Apr., 1997), pp. 756-763
7. Roukens, A., Vossen, A., Bredenbeek, P., van Dissel, J., & Visser, L. (2008). Intradermally Administered Yellow Fever Vaccine at Reduced Dose Induces a Protective Immune Response: A Randomized Controlled Non-Inferiority Trial PLoS ONE, 3 (4) DOI: 10.1371/journal.pone.0001993
8. Ann Trop Med Parasitol. 1953 Dec;47(4):381-93.  Vaccination by scarification with 17D yellow fever vaccine prepared at Yaba, Lagos, Nigeria.CANNON DA, DEWHURST F.

   Am J Hyg. 1952 Jan;55(1):140-53.  A preliminary evaluation of the immunizing power of chick-embryo 17 D yellow fever vaccine inoculated by scarification.DICK GW.

9. And I’m pretty sure I’ve seen at least one other example, but I’m blanking on the details

In the past few weeks not only did I post a short update on the DRiPs hypothesis here, but coincidentally a bunch of papers on DRiPs have also been published. I’ll probably cover some of these in more detail at some point, but here are some of the recent papers and my brief comments.

Just as a reminder: the DRiPs (“Defective ribosomal products”) hypothesis proposes that most of the peptides presented to cytotoxic T lymphocytes don’t come from the actual proteins that we normally measure — rather, the immunologically relevant peptides come from deformed and defective proteins that are mis-read and misfolded during their translation. (More explanation of DRiPs here and here; more explanation of how T cells recognize cells and where peptides come in, here.)

Jon Yewdell’s insight,¹ which is still somewhat controversial, was that defective proteins may actually be very common. Instead of being rare and abnormal events, he argued, protein production is a highly error-prone business, and a large fraction of newly synthesized proteins are broken. These defective products are very rapidly recycled into peptides and amino acids, and because of this rapid recycling they are the major source of peptides for T cell recognition.

On his original publication I had no problem with the underlying concept, but wasn’t overwhelmed by the data, and felt that there were too many counterexamples; since then he, and others, have put forward more and more examples, and I think it’s also fair to say that Jon has softened a little on the original hypothesis.² I’m more or less convinced that DRiPs are one important source of peptides, though I remain dubious that they are the only, or (and here I get very uncertain) even the major source.

Anyway, in the past few weeks, we’ve seen these papers:

• The Synthesis of Truncated Polypeptides for Immune Surveillance and Viral Evasion³

This is from Nilabh Shastri, and it’s not a big conceptual departure from some of his previous work. He’s argued for quite a while that aberrant proteins are major sources of T cell targets (see my posts here and here, for examples). Here he extends the argument to the EBNA1 protein from Epstein-Barr virus. This is a remarkably interesting protein for many reasons, one of which is that there’s reason to believe that DRiPs must be the only real source of T cell targets from EBNA1. Here, Shastri shows that in fact DRiPs (in the forms of truncated synthesis products) are in fact targets for T cells (“Thus, translation of viral mRNAs as truncated polypeptides is important for determining the antigenicity of virus proteins“). (I don’t know if it’s fair to generalize to all viral mRNAs from this very unusual protein, though.)  Very intriguingly, he also shows that DRiPs seem to be specifically blocked by EBNA1 mRNA!

Regulating production of DRiPs at the level of mRNA translation may serve as an immune evasion strategy for latent viruses. …  It is tempting to speculate that episome maintenance proteins, found in herpesviruses of various species, might have evolved to inhibit pMHC I presentation by interfering with production of DRiPs.

Is this a new viral immune evasion mechanism? And if so, how widespread is it? I know Nilabh (or someone from his lab) reads this blog occasionally, and I’d be interested in hearing their ideas on this — is it pure speculation, or do they have reason to extend the observation?

• Viral adaptation to immune selection pressure by HLA class I–restricted CTL responses targeting epitopes in HIV frameshift sequences⁴

HIV-1 frameshift inducing element

These authors looked at proteins produced by reading frame shifts from HIV.  Although HIV does a lot of frame-shifting “deliberately”, here we’re looking at frame-shifts that are (probably) not “real”.  That is, while it’s possible that some of these proteins have a biological function, for the most part they’re probably nonsense proteins, the product of incorrect selection of reading frames by the ribosome, and therefore you’d expect them to be recognized as improper proteins by the quality-control system and rapidly destroyed. In that sense they fit into the “DRiPs” concept. This fits neatly with Shastri’s previous work on frame-shifting, as well as providing modest support of the DRiPs concept.

The interesting thing here is that this paper offers evidence for large-scale immunological importance of peptides from frame-shifted proteins.  Shastri has previously shown convincing evidence that peptides derived from frame-shifted proteins can be recognized by T cells, but I always wondered if that was just a test-tube novelty. In this paper, though, Berger et al. argue that these frame-shifted potential targets show evidence of evolutionary selection, suggesting that they are recognized often enough to be a significant factor in the viral life-cycle.

• CD8 T cell response and evolutionary pressure to HIV-1 cryptic epitopes derived from antisense transcription. ⁵

And this is a very similar paper, showing the same thing for antisense-derived peptides. Like the frame-shifted proteins discussed above, these antisense proteins would probably be nonsense and rapidly degraded — defective ribosomal products, in other words — and again, there’s some evidence that these are under immunological selection, suggesting that this recognition is a real-world phenomenon.

These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity.⁵

• The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells. ⁶

This is a particularly cool paper.⁷ We know that APOBEC3G — a host protein that evolved, apparently, to provide protection against infection with retroviruses such as HIV — acts by driving hypermutation of infecting retroviral genomes. HIV resists this effect through its protein vif, which in turn drives rapid degradation of several APOBECs.

But in spite of this vif-mediated protection, it’s probably true that APOBECs still have some effect on HIV, especially very early in an infection before vif can take them out; so there’s a background of mutation in HIV driven by APOBECs. This paper shows that APOBEC-driven mutation improves T cell recognition of HIV-infected cells, and the effect is probably because the mutations force HIV to make even more defective proteins, so that there are more T cell targets. This was done in rather an artificial system (mainly by either eliminating vif altogether, or by cranking up the levels of APOBEC3G artificially), so it’s not clear how important it would be in a natural infection.

I also wonder if this argues against the notion that DRiPs are normally a big factor, because if so the background of DRiP-derived peptides should be quite high and increasing it might not be a big factor; but that’s a quantitative issue that’s hard to deal with. Still, an interesting take on antiviral effects.

• Defective Ribosomal Products Are the Major Source of Antigenic Peptides Endogenously Generated from Influenza A Virus Neuraminidase ⁸

“Drips” (Inger Taylor)

This is the paper that most explicitly tests DRiPs, which is not surprising, since it comes from Jon Yewdell himself.⁹ The paper starts with quite a fair summary of the hypothesis’s status, including some of the problems with previous experiments:

In all of these studies, we used recombinant vaccinia viruses (VVs) to express SIINFEKL-containing source Ags. It is possible that we grossly overestimated the contribution of DRiPs to Ag processing in these studies due to the use of VV to express non-VV genes. We recently showed that differences in viral translation mechanisms can greatly increase the fraction of DRiPs; expression of influenza A virus (IAV) nuclear protein by an Alphavirus vector resulted in the defective translation of >50% of nuclear protein recovered from cells. VV expression is known to modify the Ag processing pathway of some inserted viral gene products compared with their natural infection context. Further, the fusion of multiple genes to create chimeric proteins can greatly decrease the fidelity of protein synthesis or protein folding …⁸

In an attempt to get around some of these problems, they tried to come up with a more natural system.  What they built is more natural, but still is fairly artificial (as they acknowledge); still, their findings did add more support to the basic idea. (As a sign that Jon has softened his position some in the past decade, their comment “Although DRiPs are clearly a major source of antigenic peptides, it is important to recognize that peptides are also generated from natural protein turnover” is one that I think all but the most hardened anti-DRiPers would agree with; it’s coming down to a question of quantitation, of what “major” actually means, rather than absolutes.)

I still suspect that there are cases where DRiPs are critical, and cases where they’re not particularly important, and I don’t have a good sense for how many instances of each there are. My gut feeling is about half and half, but it’s not something I’d defend with my life.

1. Yewdell, J. W., Aton, L. C., and Benink, J. R. (1996). Defective ribosomal products (DRiPs): A major source of antigenic peptides for MHC class I molecules? J. Immunol. 157, 1823-1826
2. Which has made it a bit of a moving target when it comes to disproving it, unfortunately
3. Cardinaud, S., Starck, S., Chandra, P., & Shastri, N. (2010). The Synthesis of Truncated Polypeptides for Immune Surveillance and Viral Evasion PLoS ONE, 5 (1) DOI: 10.1371/journal.pone.0008692
4. Berger, C., Carlson, J., Brumme, C., Hartman, K., Brumme, Z., Henry, L., Rosato, P., Piechocka-Trocha, A., Brockman, M., Harrigan, P., Heckerman, D., Kaufmann, D., & Brander, C. (2010). Viral adaptation to immune selection pressure by HLA class I-restricted CTL responses targeting epitopes in HIV frameshift sequences Journal of Experimental Medicine, 207 (1), 61-75 DOI: 10.1084/jem.20091808
5. Bansal, A., Carlson, J., Yan, J., Akinsiku, O., Schaefer, M., Sabbaj, S., Bet, A., Levy, D., Heath, S., Tang, J., Kaslow, R., Walker, B., Ndung’u, T., Goulder, P., Heckerman, D., Hunter, E., & Goepfert, P. (2010). CD8 T cell response and evolutionary pressure to HIV-1 cryptic epitopes derived from antisense transcription Journal of Experimental Medicine, 207 (1), 51-59 DOI: 10.1084/jem.20092060
6. Casartelli, N., Guivel-Benhassine, F., Bouziat, R., Brandler, S., Schwartz, O., & Moris, A. (2009). The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells Journal of Experimental Medicine, 207 (1), 39-49 DOI: 10.1084/jem.20091933
7. I’m presenting this one on Friday in the Immunology Journal Club I run here.
8. Dolan, B., Li, L., Takeda, K., Bennink, J., & Yewdell, J. (2009). Defective Ribosomal Products Are the Major Source of Antigenic Peptides Endogenously Generated from Influenza A Virus Neuraminidase The Journal of Immunology, 184 (3), 1419-1424 DOI: 10.4049/jimmunol.0901907
9. Interestingly, it looks as if Jon has turned his attention back to influenza viruses in the past year — he cut his teeth on influenza, quite a number of years back, but it hasn’t been his main focus for a while. I guess H1N1 gave him the excuse he needed to move back that way.

Anyone who’s taken a virology class, and many who haven’t, know about “antigenic drift” and “antigenic shift”. These are usually used to explain influenza virus changes over time (although of course the same concepts apply to many other viruses). Antigenic shift refers to large, abrupt changes in the virus;² antigenic drift refers to smaller changes. Antigenic shifts are associated with pandemic influenza, as the pre-existing immune responses to influenza from previous years aren’t protective any more (because the virus has shifted away from them). Antigenic drift doesn’t let the virus escape immune control altogether, but does give the virus an advantage in infecting people — presumably, people with strong responses are still protected, but those whose exposure was maybe a few years ago, or who happened to make a weaker antibody response, to the previous virus, would be susceptible to the new virus but protected against the original. Antigenic drift happens all the time, and new drifted variants of influenza take over every year or two, which is why we need new seasonal flu vaccines on a regular basis.

What drives antigenic drift? The simple answer is that it’s driven purely by immunity. According to this notion, viruses that are resistant to being neutralized by antibody are most able to replicate and transmit to new hosts. Jon Yewdell’s group, however, has just revisited antigenic drift,¹ and propose a somewhat different model: Antigenic drift is the result of viruses cycling between immune and non-immune hosts, and it’s almost a side effect of the way the virus interacts with cells.  (Unusually for Jon, I don’t htink he coined any new and exciting acronyms for his new model.)

Model for antigenic drift selection (click for larger version)1

Normally, influenza virus doesn’t “want” to bind very strongly to its cellular target, ³ because then it’s harder for newly-formed viruses to escape from the cell (because it binds to the receptors on the way out, as well). But in the presence of neutralizing antibody, the virus needs to bind more strongly to the receptor to overcome the effects of antibody binding. Yewdell’s group argue that it’s this whip-saw effect that pushes long-term changes in antigenicity:

Thus, antigenic drift can be a by-product of Darwinian selection for mutations that optimize host cell receptor binding during influenza A virus transmission between immune (increased receptor binding) and naïve individuals (decreased receptor binding). ¹

One difference between Jon’s model and the standard concept is that with the latter, you’d expect that there would be more antigenic drift as immunity increases among the population. Yewdell’s model, though, predicts that sequential passage through immune and non-immune individuals drives antigenic drift. This actually leads to an important prediction:

In our model, antigenic drift is accelerated by sequential passage of influenza A virus between immune and nonimmune individuals, which in the human population are nearly all children. Therefore, decreasing the naïve population size by increasing pediatric influenza A virus vaccination rates will likely slow antigenic drift and temporally extend the effectiveness of influenza vaccines.¹

(My emphasis.)  They also point out that, because changes in antigenicity run in parallel with changes in receptor binding affinity, antigenic drift can itself could be pushing other changes in virus personality. That’s because the drift changes push changes in receptor binding, which in turn alters the cells to which the virus can interact; and changing the cells that the virus infects will inevitably change the nature of an infection as well. For example, does the virus best infect cells of the upper respiratory tract — leading to coughs and sniffles — or the lower respiratory tract — leading to pneumonia.

1. Hensley, S., Das, S., Bailey, A., Schmidt, L., Hickman, H., Jayaraman, A., Viswanathan, K., Raman, R., Sasisekharan, R., Bennink, J., & Yewdell, J. (2009). Hemagglutinin Receptor Binding Avidity Drives Influenza A Virus Antigenic Drift Science, 326 (5953), 734-736 DOI: 10.1126/science.1178258
2. “Changes” here mean changes in the ways the immune system responds to the virus — hence, “antigenic” changes. In practice, the changes are antibody-based changes, although in principle antigenic shift and drift could also refer to T cell-based recognition.
3. The influenza hemagglutinin, HA, protein binds to sialic acid on cells of the respiratory system.

Persons who were born before 1957 had a reduced risk of infection …  Persons who were born between 1957 and 1975 were at intermediate risk for infection. ¹

In Ontario, people over 53 years old had about 1/6 the chance² of getting the new H1N1; the those between about 33 and 53 had a little more than half the chance (odds ratios of about .15 and .42, respectively).

1. David N. Fisman, Rachel Savage, Jonathan Gubbay, Camille Achonu, Holy Akwar, David J. Farrell, Natasha S. Crowcroft, & Phil Jackson (2009). Older Age and a Reduced Likelihood of 2009 H1N1 Virus Infection The New England Journal of Medicine, 361, 2000-20001
2. I realize that odds ratios don’t quite say this, but it’s close enough

Disease-causing T cells first adhere to the inner walls of the pial vessels and then crawl in continuous contact with activated endothelial cells, most often in the opposite direction to the blood flow. …  After crossing the blood-vessel wall, the lymphocytes move along the outer surface of the vessel, encountering an array of antigens displayed by antigen-presenting cells, including macrophages. …  Last, the cells detach from the outer surface of the blood vessel and enter the spinal cord, travelling most often alongside penetrating vessels. In the spinal cord, they initiate tissue injury.²

There are a myriad of stunning videos and images.  Here’s just one video of the many, showing T cells (in green) exiting a blood vessel in the brain, and (in part 1) swimming off into the brain tissue to spread devastation and destruction (since these are autoimmune, self-reactive T cells):

1. Bartholomäus, I., Kawakami, N., Odoardi, F., Schläger, C., Miljkovic, D., Ellwart, J., Klinkert, W., Flügel-Koch, C., Issekutz, T., Wekerle, H., & Flügel, A. (2009). Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions Nature, 462 (7269), 94-98 DOI: 10.1038/nature08478
2. Ransohoff, R. (2009). Immunology: In the beginning Nature, 462 (7269), 41-42 DOI: 10.1038/462041a

Of the tools that are available or envisioned, only a highly efficacious, long-lasting vaccine would provide the degree and duration of transmission-blocking needed to achieve the simultaneous protection applied across a whole population at contiguous risk that is required to reduce and maintain R₀ < 1 for that entire area

–Plowe, C., Alonso, P., & Hoffman, S. (2009). The Potential Role of Vaccines in the Elimination of Falciparum Malaria and the Eventual Eradication of Malaria The Journal of Infectious Diseases DOI: 10.1086/646613

But:

… taking Bill and Melinda Gates’ challenge to heart and considering it seriously, we have come to the conclusion that eradication just might be possible, but only if a new set of tools are developed that focus on reducing the effectiveness of the mosquito vector. … Could a vaccine alone eradicate malaria? … A vaccine used in combination with antimalaria drugs and vector control could be quite effective in reducing the disease burden. However, eradication is a different story. We would argue that, in addition to vaccines, antimalarial drugs, and presently available vector control methods, eradication will require special tools that we have yet to develop.

–Miller, L., & Pierce, S. (2009). Perspective on Malaria Eradication: Is Eradication Possible without Modifying the Mosquito? The Journal of Infectious Diseases DOI: 10.1086/646612

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

[W]e have estimated that natural selection drives twice as much change in immune-related proteins as in proteins with no immune function. Interestingly, the rate of adaptation is also more variable among immunity genes than among other genes in the genome, with a small subset of immunity genes evolving under intense natural selection. We suggest that these genes may represent hotspots of host–parasite coevolution within the genome.

–Obbard, D., Welch, J., Kim, K., & Jiggins, F. (2009). Quantifying Adaptive Evolution in the Drosophila Immune System PLoS Genetics, 5 (10) DOI: 10.1371/journal.pgen.1000698

(This is particularly interesting to me because I’m trying to look at co-evolution between pathogens and immunity myself.  I’ve been tentatively suggesting that adaptive immune components (co)-evolve faster than innate immune components; of course, Drosophila only have innate immunity, so this paper suggests that the innate immune system also evolves rapidly.  That’s not unexpected, and doesn’t disprove my hypothesis, but it’s interesting anyway.  Also, there are some techniques in here I might be able to make use of.)

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

In the past few years, we’ve learned a little more, quantitatively, just how dramatic this expansion phase is. Delicate work has established that there are maybe 20 to 1000 potentially-reactive T cells in a mouse, before infection (see my discussion here and here). Those few cells are the precursors of the huge numbers of T cells a week or so after infection.

If you think about it, it’s pretty remarkable that these few T cells, hidden within millions upon millions of other, irrelevant, T cells, ever get the signal to divide. That signal is carried by specialist antigen-presenting cells, most often dendritic cells. A simplified overview goes something like this: A dendritic cell is hanging out somewhere in the body — let’s say in the skin. It’s constantly filter-feeding, sampling the surrounding environment. Mostly, this surrounding environment is innocuous; there are only normal self antigens in it. If the DC finds evidence of an infection, like, say, viral RNA, then it grinds into action; it migrates to a local lymph node and shows the local T cells everything it (the DC) has been exposed to over the past 24 hours or so. Early in an infection, there usually aren’t a lot of pathogens present, and so there aren’t very many of these activated DC; maybe a few hundred or a thousand.

Meanwhile, the naive T cells are also roaming around, from lymph node to lymph node, scanning as many DC as they can. If there’s nothing they recognize in the DCs of one lymph node, off they go to the next for more scanning.

So for a T cell to get the go signal, a tiny number of specific T cells (behaving identically to the vast number of irrelevant T cells) have to make contact with a tiny number of specific DC (hidden within the vast expanse of all the body’s lymphoid tissue), just by randomly bumping into each other.

Now, here’s a question: When you get that large pool of activated T cells a week after infection, are these the product of all (let’s say) 500 precursors, or are they all the progeny of one or two lucky precursors that managed to bump into the right DC? In other words, how efficient is the bumping-into-DC process? It’s remarkable enough that it works at all; could it, even more remarkably, be so efficient that all the possible precursors manage to find a partner in the first day?

Amazingly enough, the answer is yes, all the precursors do find a partner. A paper from Ton Schumacher’s group² has showed that there are virtually no wallflowers. Recruitment into the activated state is close to perfect (they demonstrate 95%). They go on to calculate how many random interactions you need, to get this level of recruitment, and suggest that there have to be over 50 million interactions to get this level of recruitment — but this is still in the right ballpark from what we know about rate of interactions:

Assuming that naïve antigen-specific CD8+ T cells are present at a frequency of ~1:100,000 within a CD8+ T cell pool of ~20 x 10⁶ cells, it would require around 59 x 10⁶ T-DC interactions to achieve 95% recruitment, a number that is largely independent of variations in precursor frequency within the physiological range. It has been estimated that DCs are able to interact with at least 500 different T cells/hour; thus, a pool of <2000 antigen-presenting DCs could suffice to achieve this near-complete recruitment. ²

A couple of groups have made movies of T cell/DC interactions, and when you look at those the figures seem more plausible.  Here³ is a movie from the Cahalan lab.⁴  (Go to their lab page for more fascinating immunology movies, by the way.) This is two-photon microscopy, taken in the lymph node of a live mouse.  DC are green, T cells are, hmm, sort of an orangey red.  The left panel shows T cells interacting with DC that don’t have the appropriate antigen; the T cells charge in, take a quick look at the DC, bounce off, and move on to the next one.  (This movie is time-compressed, of course; we’re looking at a couple of hours here.)  On the right, we see DC that do have the appropriate target for the T cells.  Here the T cells have bumped into the DC and immediately stopped looking further; they just sort of hang with the DC, soaking up the information, and preparing to move off into the activated T cell program.

The frenetic action on the left gives a sense of the rate of interaction, and 500 T cells per hour doesn’t seem too far off.

1. And then, as part of the same program that triggered their massive expansion, the cast majority of the expanded T cells die off again, restoring the immune system almost to the original status quo.
2. van Heijst, J., Gerlach, C., Swart, E., Sie, D., Nunes-Alves, C., Kerkhoven, R., Arens, R., Correia-Neves, M., Schepers, K., & Schumacher, T. (2009). Recruitment of Antigen-Specific CD8+ T Cells in Response to Infection Is Markedly Efficient Science, 325 (5945), 1265-1269 DOI: 10.1126/science.1175455
3. If I’ve embedded this properly
4. Miller, M. (2002). Two-Photon Imaging of Lymphocyte Motility and Antigen Response in Intact Lymph Node Science, 296 (5574), 1869-1873 DOI: 10.1126/science.1070051