Mystery Rays from Outer Space

Does the immune system control tumors?

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[↩]

Blood vessels, colorectal cancer

Although I usually talk about reduced immune responses in relation to tumor progression, I’ve also mentioned the possibility that chronic inflammation — excessive immune response –  may enhance tumor progression as well. For example, chronic hepatitis B infection leads to about a 10-fold increase in the risk of liver cancer, probably because of the chronic inflammation associated with the virus. ¹ At least one reason for the increased risk is that inflammation produces reactive oxygen and nitrogen species (RONS). RONS are toxic to microbes, but not surprisingly they’re also somewhat toxic to the host cells as well. In particular, RONS are mutagenic.

At any rate, that’s been the proposed reason for the increased risk, but this apparently hadn’t been formally tested. (It’s not a field I follow intently, so I am taking someone else’s word on that; but I don’t know of any tests.) A paper in Journal of Clinical Investigation now has looked at this specifically², and though it’s not directly related to viral immunity the message is probably relevant in that context as well. The authors asked what happens when chronic inflammation takes place in mice that can’t efficiently repair the DNA damage that RONS induces. (These are mice that lack a particular DNA repair enzyme, alkyladenine DNA glycosylase (Aag).)

Helicobacter pylori

If the hypothesis is correct, then the wild-type and the mutant (repair-deficient) mice should both be okay with acute inflammation, but the mutant mice should have increased risk of cancer with chronic inflammation. Using a chemical to induce gastrointestinal inflammation, that’s pretty much what happened; there were more cases of colorectal cancer in the mutant mice that couldn’t repair RONS-induced DNA damage. ³Â  However,this isn’t due to a globally higher risk of tumors.  The mutant mice didn’t have any different rate of tumors in a different model of carcinogenesis (crossed to a tumor-prone strain of mice), implying that it is specifically chronic inflammation that’s the problem.

They then moved the experiment into clinical territory by infecting the mice with Helicobacter pylori, the stomach ulcer bacterium. Humans infected with H pylori have chronic inflammation of the gastrointestinal tract, of course, and they also have increased risk of gastric cancer.⁴ The mutant mice didn’t actually develop tumors in this experiment, but they did have much more severe gastric lesions (”histopathologic lesions that were markers of increased predisposition to, or were precursors to, gastric cancer“).

So reducing the ability to clean up after RONS doesn’t alter the normal risk of cancer, but does increase the risk when chronic inflammation is present. In an interesting conclusion, the authors suggest that it might be useful to look at variation in these DNA repair enzymes as a marker for tumor risk in several diseases:

This may be particularly important for gene-environment interactions with states of chronic inflammation or with other conditions known to increase oxidative stress such as metal storage diseases, heavy metal exposure, smoking, and chronic infection.

1. For a review: de Visser, K.E., Eichten, A., Coussens, L.M. (2006). Paradoxical roles of the immune system during cancer development. Nature Reviews Cancer, 6(1), 24-37. DOI: 10.1038/nrc1782[↩]
2. Meira, L.B., Bugni, J.M., Green, S.L., Lee, C., Pang, B., Borenshtein, D., Rickman, B.H., Rogers, A.B., Moroski-Erkul, C.A., McFaline, J.L., Schauer, D.B., Dedon, P.C., Fox, J.G., Samson, L.D. (2008). DNA damage induced by chronic inflammation contributes to colon carcinogenesis in mice. Journal of Clinical Investigation DOI: 10.1172/JCI35073[↩]
3. Actually, they say it’s exactly what happened, but I’m a little puzzled by their statistics; unless I’m missing it, though, there’s not enough information included in the paper for me to repeat the analysis.[↩]
4. Helicobacter pylori infection and the pathogenesis of gastric cancer: A paradigm for host-bacterial interactions. D. McNamaraa and E. El-Omar. Digestive and Liver Disease (2008) 40:504-509 doi:10.1016/j.dld.2008.02.031 [↩]

Although 750-odd tumor antigens may seem like quite a few potential vaccine targets, it’s really not so much when you’re dealing with billions of individual tumors; and so when designing a tumor vaccine, you may have to make some compromises. The peptide may bind to the MHC class I with low affinity, for example, making it relaitvely non-immunogenic. Several groups who are working on tumor vaccines have tried to work around this problem by optimizing tumor antigens in various way, hoping to boost the immunogenicity while retaining the specificity of the peptide. This has often seemed to work quite nicely, cranking up immune responses significantly while keeping the response focused on the tumor. Nevertheless, a recent study¹ suggests that this may not be a good idea.

It has been suggested² that cancer antigens tend to be poor MHC binders. That would mean that the peptide falls out of the MHC complex relatively rapidly and becomes invisible to T cells, so that to keep a certain level of target on the cell surface, you’d need to start with much more; in other words, the peptide would be less immunogenic. (This has been explicitly shown for a number of tumor epitopes, but as far as I know has not been globally demonstrated. It occurs to me that the Immune Epitope Database [IEDB]   may have enough information to at least make a start at that analysis; maybe I’ll take a run at it, in whatever of my  free time isn’t taken up by playing baseball with my fanatic son.)

If the natural peptide doesn’t bind stably to MHC, perhaps an analog peptide — a peptide with a slightly different amino acid sequence — can be made, with the same T cell recognition properties, that does bind well; and this analog could be used for immunization. That’s just what has been done in a number of clinical trials,³ and the results have actually looked good; for example, mice immunized with an analog peptide of a melanoma tumor antigen generated far more T cells than with the natural antigen. ⁴

But bigger is not always better, and now Speiser et al. have examined the effects of an analog peptide qualitatively as well as quantitatively. Again using a melanoma antigen, they compared  the immune response to a natural and an analog peptide vaccination. As with other studies, the analog peptide induced more T cells; about twice as many. But the quality⁵ of the T cells induced by the natural peptides was much better, to the point that the less abundant natural response, was more effective in its anti-tumor function than the more abundant response induced by the analog peptides.

At first, it seems paradoxical that the “less immunogenic” natural peptide induced more strongly functional T cells. … CD8 T cells must be able to recognize low amounts of viral peptide antigen for protection. More recently, in vivo experiments in mice showed that the peptide concentration used for DC labeling and priming inversely correlated with the avidity of TCRs of memory cells. Thus, one may conclude that vaccination should be done with low peptide doses and/or peptides with low HLA binding stability (provided that one can still elicit a reasonably strong T cell response).

(My emphasis.)  This is actually strikingly reminiscent of some of the recent work on viral — especially HIV — immune responses, where T cell quality (induction of “multifunctional” T cells) seems to be more important than maxing out the number of T cells. ⁶.  I guess it’s not surprising that the anti-cancer and anti-viral responses are similar in this, as they are in many other ways.

1. Speiser, D.E., Baumgaertner, P., Voelter, V., Devevre, E., Barbey, C., Rufer, N., Romero, P. (2008). Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen. Proceedings of the National Academy of Sciences, 105(10), 3849-3854. DOI: 10.1073/pnas.0800080105[↩]
2. For example: Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance. Zhiya Yu, Marc R. Theoret, Christopher E. Touloukian, Deborah R. Surman, Scott C. Garman, Lionel Feigenbaum, Tiffany K. Baxter, Brian M. Baker, and Nicholas P. Restifo. J Clin Invest. 2004 August 16; 114(4): 551-559. doi: 10.1172/JCI200421695. [↩]
3. Speiser et al. cite a half dozen instances; I won’t parrot them[↩]
4. Parkhurst et al. (1996) Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified HLA-A*0201-binding residues. J Immunol 157:2539-2548. [↩]
5. Quality in this case means functional ability to deal with the cancer; it includes things like activatability, amount of cytokine production, and amount of lytic proteins produced[↩]
6. For example: Induction of multifunctional human immunodeficiency virus type 1 (HIV-1)-specific T cells capable of proliferation in healthy subjects by using a prime-boost regimen of DNA- and modified vaccinia virus Ankara-vectored vaccines expressing HIV-1 Gag coupled to CD8+ T-cell epitopes. Goonetilleke N, Moore S, Dally L, Winstone N, Cebere I, Mahmoud A, Pinheiro S, Gillespie G, Brown D, Loach V, Roberts J, Guimaraes-Walker A, Hayes P, Loughran K, Smith C, De Bont J, Verlinde C, Vooijs D, Schmidt C, Boaz M, Gilmour J, Fast P, Dorrell L, Hanke T, McMichael AJ. J Virol. 2006 May;80(10):4717-28. and references therein.[↩]

… it may be important to readdress the therapeutic management of different cancers, given the idea that chemotherapy should elicit an immune response.  …  it could be important to preserve the sentinel lymph node rather than remove it systematically for disease staging purposes. Indeed, the sentinel lymph node constitutes the privileged site of antigen priming, in which the presence of activated DCs (expressing DC-LAMP - a protein only expressed in mature DCs) constitutes a positive prognostic marker, at least in melanoma.

–The anticancer immune response: indispensable for therapeutic success?
Laurence Zitvogel, Lionel Apetoh, François Ghiringhelli, Fabrice André, Antoine Tesniere and Guido Kroemer. J. Clin. Invest. 118(6): 1991-2001 (2008). doi:10.1172/JCI35180.

Natural killer cells were originally identified as cells that spontaneously killed cancer cells. It’s been a bit surprising, then, that there’s been relatively little direct evidence that NK cells protect against spontaneous cancer.

For example, there was the study I talked about some time ago, looking at tumors in equilibrium with the immune system. There, the authors treated mice with a  carcinogen, waited until tumors stopped arising, and immunosuppressed the tumor-free survivors. ¹ In most of these apparently cancer-free animals, immune suppression resulted in tumors appearing within a few weeks. This showed that the immune system can control tumors.

The interesting part (at least, the interesting part as far as NK cells are concerned) was that treatment with anti-NKG2D did not let tumors grow out; whereas shutting down T cells (with anti-CD4/anti-CD8) or interferon (with anti-IFN) did let the tumors reappear. NKG2D is an important receptor for NK cells, so the implication was that NK cells were not keeping the tumor in check, but T cells were.

Now, however, a similar set of experiments has shown that’s not necessarily true — NK cells probably are important in controlling some tumors. The paper is
Guerra, N., Tan, Y. X., Joncker, N. T., Choy, A., Gallardo, F., Xiong, N., Knoblaugh, S., Cado, D., Greenberg, N. R., and Raulet, D. H. (2008). NKG2D-Deficient Mice Are Defective in Tumor Surveillance in Models of Spontaneous Malignancy. Immunity 28, 571-580. DOI: 10.1016/j.immuni.2008.02.016

David Raulet’s group has been one of the leaders in NK cell research, and as far as I know they are the first to have made a knockout mouse lacking NKG2D. The mice are normal and happy and actually have normal numbers of NK cells that are functional. NK cells notoriously have many ligands, which is one of the reasons it took longer to figure out NK receptor/ligand interactions than for T cells, and presumably there are enough alternatives to NKG2D that NK cells can get whatever signals they need during development. However, of course, none of the ligands for NKG2D triggered NK cells.

Rather than wait for truly spontaneous tumors (which are rare enough even in mice that you need very large numbers of mice to figure out what’s going on) they crossed the NKG2D -/-mice with a couple of transgenic lines that are highly cancer-prone. They also tried treating the mice with carcinogens, as was done in the Koebel et al study I mentioned earlier.

The carcinogen-treated NKG2D knockout mice got no more tumors than did wild-type mice — so that’s exactly consistent with the previous experiment. However, the “spontaneous” tumor transgenic models showed a big difference. The NKG2D knockouts had much earlier, more aggressive tumors than did the wild-type mice.

As well as evidence for cancer equilibrium, the Koebel et al paper showed evidence for immunoediting. ² That is, tumors that grow in the presence of a healthy immune system are resistant to the immune response — they have been selected for immune invisibility or resistance. By comparison, tumors that grow in immune deficient mice are much more immunogenic — they haven’t had to develop immune resistance.

In one of the two transgenic systems, the NKG2D knockout mice showed the same thing: Their tumors were more likely to have NKG2D ligands than the wild-type mice. “These data suggest that NKG2D-dependent immunoselection (or editing) favors loss of NKG2D ligands on early-arising, aggressive tumors.”

But in the other tumor system no such evidence was seen; NKG2D ligands were just as prevalent:

There was no indication in this survey that expression of NKG2D ligands was selected against in Klrk1+/+ mice, despite the clear evidence that NKG2D-mediated surveillance is operative for these lymphomas. These data suggest that evasion of NKG2D-mediated surveillance by Eμ-myc-induced lymphomas occurs by mechanisms that do not depend on loss of NKG2D ligands.

So it seems that NK immune surveillance is much more complicated than the (already very complicated) T cell immune surveillance of tumors:

Taken together, these data suggest that the role of NKG2D-dependent surveillance differs in the three types of tumors studied here. In the case of early-arising prostate carcinomas in TRAMP mice, many of the tumors are eliminated, and the few that are not eliminated evade surveillance by extinguishing expression of NKG2D ligands. In the case of Eμ-myc lymphomas, it appears that the emerging tumors are mostly NKG2D sensitive, but a fraction of tumors escape NKG2D surveillance without losing NKG2D ligands. … The final category is represented by late-arising prostate carcinomas, which appear to be generally refractory to NKG2D-dependent surveillance.

But the bottom line is that NK cells do control some tumors. That’s not a surprise, because it’s pretty much been the assumption for a long time, but it’s reassuring to get evidence for it.

1. Koebel, C. M., Vermi, W., Swann, J. B., Zerafa, N., Rodig, S. J., Old, L. J., Smyth, M. J., and Schreiber, R. D. (2007). Adaptive immunity maintains occult cancer in an equilibrium state. Nature 450, 903-907.[↩]
2. Of course, this wasn’t the first evidence for immunoediting.[↩]

A while ago I talked about a paper that demonstrated that cancers establish a long-term equilibrium with the immune system. That paper provided a formal demonstration of a concept that was already widely accepted — that cancers can arise and persist for a long time in balance with the immune system, so that the immune system isn’t able to completely eliminate the cancerous cells, but the tumor is not able to grow or spread, either. Evidence in humans shows that this equilibrium can persist for many years. In some cases, the tumor may eventually mutate to a form that can more completely avoid the immune system, and those tumors become clinically detectable. In other cases, perhaps, the immune system may finally destroy all vestiges of the tumor, and the person won’t ever know that he was carrying the potentially-cancerous cells; or the balance may persist for a lifetime, until the person dies of something altogether different.

There was an interesting implication to that concept that I had overlooked, because it doesn’t fit with the conventional understanding of cancer development. A paper from last month¹ proposes a new and rather troubling model.

People rarely die of a primary tumor; the direct cause of death is usually metastatic cancer. The prevailing models of cancer development suggest that metastasis is a late event in cancer development. (I believe this concept was first articulated by Fidler in 1978,² although in retrospect this paper only argues that metastasis is the product of a subset of selected cells from the tumor — the concept that a tumor is not a homogenous entity — not that the selection process that leads to the metastatic subset must necessarily occur late in the cancer’s progression.) Hanahan and Weinberg, in their immensely influential 2000 paper “The Hallmarks of Cancer”,³ discussed “Tissue Invasion and Metastasis” as the sixth and final “acquired capabilities” of a tumor, and show metastasis as the final event in four of the five pathways they illustrated, and as the penultimate event in the other one (see the figure to the right here; click for a larger version).

However, there’s also some evidence that metastasis isn’t necessarily a very late event in cancer development. For example, Engel et al. worked out the point at which metastases from breast cancers must have occurred, based on their growth rates, and determined that metastases must often happen quite early in the course of the tumor: ⁴

Our hypothesis of an early MET [metastasis. IY] initiation questions the morphological and genetic correlation between the primary tumour and MET, because the primary tumour at the time of dissemination would have been much smaller and probably had prognostically favourable characteristics.

The paper I’m talking about here,¹ from Hüsemann et al, takes this argument a couple of steps further. Not only can metastases arise very early in the course of cancer development, they say, metastases can even arise before the cells are fully malignant. Using a mouse mammary carcinoma model in which mammary tumors arise in a predictable fashion, they were able to detect the premalignant mammary cells in the bone marrow as early as 4 weeks - before outright tumors had arisen in the mammary tissue itself — and these early-spreading cells were able to cause cancer in irradiated (immune-deficient) mice after bone marrow transplant.

One provocative finding of our study is that, in mouse models of breast cancer, large tumors seed neither more nor genetically further-advanced cancer cells than do small lesions… Thus, the ability of metastatic dissemination does not appear to be the result of selection of tumor cells within the tumor. Rather, the data suggest that tumor cells disseminate early and will be selected for outgrowth at distant sites.

(My emphasis.)

If metastases spread very early, then why do we even see “primary tumors”? Why don’t the metastases arise at the same time as the “primaries”, or even earlier — so that all we see is a metastatic cancer with no primary tumor? (In fact, this is exactly what is seen in a lot of tumors. Some 5 - 10% of tumors present as metastases with an unknown primary tumor. ⁵ However, the question still applies to the remaining 90-95% of tumors.)

The most likely explanation is that there is some growth restriction on metastases. For example, perhaps the primary tumor secretes growth factors systemically, which permits the metastases to expand. Or — and this is a possibility the authors didn’t really discuss, so I’m not sure if there’s some obvious flaw that I’m missing — the immune system may hold the small tumors in check for a while until the metastases manage to evade equilibrium with the immune system. I wonder if this could be a regulatory T cell (TReg) effect, in which the primary tumor is able to expand and then conditions infiltrating lymphocytes to become TRegs; we know that TRegs circulate, so perhaps these circulating TRegs then shut down the ongoing response to the metastases as well, and allow them to grow out.

In any case, this observation (assuming it holds up) changes some of the concepts of cancer therapy, I think. If the metastases are already out there, then some of the rationale for removing the primary tumor is gone. On the other hand, there’s a potential opportunity there, too: If something prevents metastases from growing out and causing a problem for a long time, perhaps that something can be harnessed and used to prevent outgrowth for a lifetime.

1. Yves Hüsemann, Jochen B. Geigl, Falk Schubert, Piero Musiani, Manfred Meyer, Elke Burghart, Guido Forni, Roland Eils, Tanja Fehm, Gert Riethmüller and Christoph A. Klein. (2008). Systemic Spread Is an Early Step in Breast Cancer. Cancer Cell, 13(1), 58-68. DOI: 10.1016/j.ccr.2007.12.003[↩][↩]
2. Fidler, I. J. (1978). Tumor heterogeneity and the biology of cancer invasion and metastasis. Cancer Res 38, 2651-2660.[↩]
3. Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57-70.[↩]
4. Engel, J., Eckel, R., Kerr, J., Schmidt, M., Furstenberger, G., Richter, R., Sauer, H., Senn, H. J., and Holzel, D. (2003). The process of metastasisation for breast cancer. Eur J Cancer 39, 1794-1806.[↩]
5. van de Wouw AJ, Janssen-Heijnen MLG, Coebergh JWW, Hillen HFP (2002) Epidemiology of unknown primary tumours; incidence and population-based survival of 1285 patients in Southeast Netherlands, 1984-1992. European Journal of Cancer 38.:409-413 [↩]

A while ago, talking about tumor development, I said that “tumors normally develop ways to avoid immunity.” It’s that ability to evade the immune system that allows tumors to escape from the equilibrium phase, when they’re a mere harmless handful of cells held in check by the immune system, and become outright clinically-detectable tumors.

How do tumors avoid immunity?

There are many ways. Remember that tumors are not like viruses; each tumor arises de novo and has only its host’s lifespan in which to evolve. It has no connection to other tumors of the same kind; there is no evolutionary linkage. All human cytomegaloviruses have a common ancestor, but no two colon cancers have the same ancestor. ¹ That means that each tumor must find its own solution to every problem rather than relying on its ancestors’ solutions. (And if most colon carcinomas, say, adopt the same solution, we have to look at what factors make that solution particularly accessible to, or appropriate for, that particular type of cancer.)

That said, there are some mechanisms that are very widely used by tumors to evade the immune system, and regulatory T cells (TRegs) are one of them. That’s probably at least in part because TRegs’ natural functions include suppressing immune responses to self antigens and reducing inflammation in chronic exposure to antigen. It’s relatively easy to get the TRegs to do their job a little more enthusiastically.

(TRegs are T cells that specifically down-regulate immune responses; without TRegs, immune responses explode and cause massive damage. People without TRegs have terrible, usually fatal, autoimmune and inflammatory disease, so you don’t want to just blithely eliminate them to get a “better” immune response. )

It was suggested nearly 30 years ago that TRegs contributed to tumor growth,² but the whole I-J fiasco set the field back a long way, and it wasn’t until relatively recently that the questions were revisited. ³ It’s now pretty clear that, in fact, TRegs are often abundant in tumors, and actively shut down immune responses to the tumors. For example, it’s been shown recently that tumors with relatively more TRegs have a worse prognosis:⁴

In patients with undesirable outcome, the balance is tipped in favor of Tregs (high Tregs and low activated CTLs), whereas in patients with relatively desirable outcome, the balance is tipped toward effector T cells (low Tregs and high activated CTLs).

Eliminating TRegs from mice with experimental tumors caused rejection of the tumors,⁵ so as you’d expect, there’s a lot of interest in this sort of approach to cancer therapy.

Relevant previous posts
Cancer:
• … and immune escape
• … and TLRs
• … and immunity
• … the Three E’s of
• … and equilibrium
Regulatory T cells:
• … and the I-J story
• … and persistent viruses
A small step forward was described recently in PNAS. ⁶ “Classic” TRegs are antigen-specific; they recognize antigen just as do T Helper cells, but instead of responding by stimulating immune responses, the TRegs respond to their antigen by suppressing local responses. There are also non-antigen-specific cells that act something like TRegs (though not as effectively). This paper shows that patients with melanoma have circulating (not just tumor-infiltrating) TRegs that are specific for tumor antigens.

This is a nice little paper, but I don’t think it represents a very large or surprising advance.⁷ It’s already known that tumor-infiltrating TRegs can be tumor-antigen-specific;⁸ it’s known that patients with melanoma have increased numbers of circulating TRegs;⁹ and it’s also been shown that tumor-specific T cells are in circulation in patients with melanoma. ¹⁰ So this (as far as I can see) extends several prior observations, but in an unsurprising direction.

Also, unless I’m missing something (which wouldn’t be new), I don’t see just how knowing or identifying circulating TReg antigens will help with treatment or prognosis. One possibility that occurs to me is that these circulating TRegs may help protect metastases from rejection. If the TRegs were all infiltrating the tumor, then perhaps the immune system would be able to deal with the small number of metastatic cells that are spreading throughout lymphatics and so on, whereas if TRegs are also circulating then the metastatic cells might be protected. I’m not entirely sure about the actual relevance of this, because even if TRegs were only tumor-infiltrating I think there would be plenty of opportunity to tolerize tumor-specific T cells.

Still, it’s always important to understand the system.

Just as a final cautionary note: As I said up at the top, tumors are all different. Different classes of tumors may be particularly able to move along certain immune evasion pathways because of their underlying characteristics. For example, inflammatory infiltrate within some tumors is a good sign¹¹ (presumably because it implies that there is an active immune response against the tumor), yet in other classes of tumor the same kind of infiltrate is a bad sign, perhaps because it indicates TReg infiltration and a suppressive environment. Even within classes of tumors there are inevitably variations. It may not be possible to develop universal rules for tumor immune treatment; but it may be possible to find some useful guidelines.

1. With bizarre exceptions like transmissible canine veneral tumor and Tasmanian Devil tumor. [↩]
2. Berendt, M.J. & North, R.J., 1980. T-cell-mediated suppression of anti-tumor immunity. An explanation for progressive growth of an immunogenic tumor. The Journal of experimental medicine, 151(1), p.69-80.[↩]
3. A nice review: Zou, W., 2006. Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol, 6(4), p.295-307. [↩]
4. Gao, Q. et al., 2007. Intratumoral Balance of Regulatory and Cytotoxic T Cells Is Associated With Prognosis of Hepatocellular Carcinoma After Resection. J Clin Oncol, 25(18), p.2586-2593. DOI: 10.1200/JCO.2006.09.4565 [↩]
5. Shimizu, J., Yamazaki, S., & Sakaguchi, S., 1999. Induction of Tumor Immunity by Removing CD25+CD4+ T Cells: A Common Basis Between Tumor Immunity and Autoimmunity. J Immunol, 163(10), p.5211-5218.
   and
   Viehl, C. T., Moore, T. T., Liyanage, U. K., Frey, D. M., Ehlers, J. P., Eberlein, T. J., Goedegebuure, P. S., and Linehan, D. C. (2006). Depletion of CD4+CD25+ regulatory T cells promotes a tumor-specific immune response in pancreas cancer-bearing mice. Ann Surg Oncol 13, 1252-1258.[↩]
6. Vence, L. et al., 2007. Circulating tumor antigen-specific regulatory T cells in patients with metastatic melanoma. Proceedings of the National Academy of Sciences, 104(52), p.20884-20889. [↩]
7. I mention it here mainly because it reminded me that this is a subject I wanted to talk about.[↩]
8. see Wang, R., 2006. Functional control of regulatory T cells and cancer immunotherapy. Seminars in Cancer Biology, 16(2), p.106-114. for a review[↩]
9. McCarter, M.D. et al., 2007. Immunosuppressive dendritic and regulatory T cells are upregulated in melanoma patients. Annals of surgical oncology, 14(10), p.2854-60.[↩]
10. For example, Michalek, J. et al., 2007. Detection and Long-Term In Vivo Monitoring of Individual Tumor-Specific T Cell Clones in Patients with Metastatic Melanoma. J Immunol, 178(11), p.6789-6795.

    and Bioley, G. et al., 2006. Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers. Journal of immunology 177(10), p.6769-79.[↩]

11. For example, Eerola AK, Soini Y, Paakko P. A high number of tumor-infiltrating lymphocytes are associated with a small tumor size, low tumor stage, and a favorable prognosis in operated small cell lung carcinoma. Clin Cancer Res. 2000; 6: 1875-1881[↩]

Oncolytic VSV (gold) infecting lung tumors1

Oncolytic viruses are a concept I’d like to be more excited by than I am.² It’s an idea that seemed really exciting when I first came across it, but the more I thought about it the more dubious I was. But a recent paper helps me feel better about at least two of my worries.

The concept is a straightforward one. Viruses are good at killing cells.³ Why not have them infect cells that we want to die? That would be, for example, cancer cells. So all you need to do is find or make a virus that only grows in cancer cells, and you’re cured. Simple! Tomorrow we’ll fix global warming!

There’s the obvious problem with this: How can you find (or make) a cancer-specific virus? In principle the answer is the same as with chemotherapy; you use the ways cancer cells are different from normal as targets. This isn’t as hard as you might think. Lots of the things that make cancer cells cancerous are similar to the things viruses like. Viruses often drive infected cells into a cancer-like state that is more hospitable to the virus — friendly to nucleic acid replication, replication, unresponsive to death signals, independent of the signals that normally regulate growth. So lots of viruses are already kind of pre-adapted to replicate well in cells with a cancerous phenotype, and it doesn’t take all that much tweaking to make them adapted to only replicate well in cancer cells.

(After writing this post, it occurred to me that this is actually topical! I don’t usually do the topical blog post thing, but the background in “I Am Legend” has an anti-cancer virus, isn’t it? I haven’t seen it myself, with the grant-writing and the teaching and the two little kids,⁴ but have I actually tied a current entertainment topic into “Mystery Rays”? Fame and fortune is certain to come my way!)

Oncolysis through the ages

The first runs at this technique that I knew of⁵ used mutant herpesviruses,⁶ but I think that much of the buzz came from work with defective adenoviruxes, especially the ONYX-015 virus.⁷ The approach here was based on the observation that adenoviruses (like many other viruses) normally inactivate p53 during infection. p53 is a multifunctional growth regulator that is very often also inactivated in cancers, for the same reason as viruses like to inactivate it:it oten triggers death in cells with unchecked growth. Adenoviruses lacking the gene that inactivates p53 (their E1B gene) can only efficiently infect cells lacking p53 — which would usually be, of course, cancer cells.

As well as herpesviruses and adenoviruses, though, all sorts of other viruses have been used.⁸ One interesting approach is vesicular stomatitis virus. This is a very, very innocuous virus in normal people, partly because VSV is extremely sensitive to interferon. (VSV is used in bioassays for interferon release, because even tiny amounts of interferon completely block the virus’s replication.) So which kind of cells often aren’t responsive to interferon? Right; cancer cells, as part of their own immune evasion pathway, frequently disable their interferon responses. VSV doesn’t infect normal cells, but does infect, and kill, tumor cells.⁹

Questions and (maybe) answers

Anyway, the first question, of specificity, is more or less under control.¹⁰ Three questions that had made me rather dubious about the concept, though, still remained:

1. Getting the virus to the tumor …
2. Especially in the face of an immune response.
3. Killing all of the cancer cells, not a mere 99% of them (from which the cancer will rapidly recover).

Malignant melanoma cells in lymph node

A paper in Nature Medicine¹¹ offers encouragement on all of those.

They used VSV as their cancer killer, and their twist here was to deliver it by loading it onto T cells. T cells naturally traffic to lymph nodes, and quite a few tumors metastasize through lymph nodes; the T cell therefore acts as a ferry to deliver its deadly viral cargo to the metastasizing tumor. (The goal here was not to clear the primary tumor, but to prevent metastases, which are often the major problem.  However, they did see some effect on the primary tumor, too, in some cases.) When it reaches the lymphoid tissue, it delivers the passenger virus to the cancer cells, the only ones that the VSV can productively infect (since the cancer cells are the only ones that have mutated their interferon pathway). This is an interesting idea, though limited in this form — I wonder about using antigen-specific T cells instead, to target the virus to a specific site — and it seemed to work quite well.

The two more interesting points to me were kind of peripheral to their main point. First, they find that once the virus killed some cancer cells, there was anti-tumor protection even after the virus was all cleared, and this was probably because of the immune response,¹² which was triggered by the cell death initially caused by the virus:

In vivo tumor cell purging resulted both from direct viral oncolysis by virus released from the T cell carriers and from the priming of protective antitumor immunity, which prevents repopulation by further waves of cells metastasizing from the primary tumor.

– just as described in the paper by Apetoh et al¹³ that I talked about here. The authors suggest that because the cancer metastases are being killed in the lymph nodes, rather than in the bulk of the tumor (which is generally a highly immunosuppressive environment) the immune response was more efficient. That starts to get past my concern #3 above, because it offers multiple attacks on the tumor, not just the virus.

The other point is that the virus could reach the cancer reasonably well even in the face of an anti-viral immune response; the trick was to use just enough virus to kill the tumor cells, without getting enough on the T cells to trigger an immune response:

In virus-immune mice, T cells loaded with large amounts of VSV (MOI 1 or 10) could not keep DLNs or spleens free of tumors. However, T cells loaded with fewer viruses (MOI 0.1) still protected even virus-immune animals from tumor colonization of the DLN and spleen

The data are still very preliminary and inconclusive, but certainly it’s a step in the right direction, and I feel better about this whole approach than I did before reading the paper.

1. Carrier Cell-based Delivery of an Oncolytic Virus Circumvents Antiviral Immunity. Anthony T Power, Jiahu Wang, Theresa J Falls, Jennifer M Paterson, Kelley A Parato, Brian D Lichty, David F Stojdl, Peter A J Forsyth, Harry Atkins and John C Bell. Molecular Therapy (2007) 15, 123-130. [↩]
2. That sentence needs a road map, but you got here eventually, didn’t you.[↩]
3. At least, lytic viruses are.[↩]
4. And the running and the screaming and the monkeys in the hair[↩]
5. I realize now, though, that the concept arose long before that, apparently in the 1950s. For example: Love R, Sharpless GR. Studies on a transplantable chicken tumor, RPL-12 lymphoma. II. Mechanism of regression following infection with an oncolytic virus. Cancer Res. 1954 Oct;14(9):640-7.http://dx.doi.org/10.1126/science.1851332 though I don’t know much about those studies other than the titles [↩]
6. Experimental therapy of human glioma by means of a genetically engineered virus mutant. Martuza RL, Malick A, Markert JM, Ruffner KL, Coen DM. Science. 1991 May 10;252(5007):854-6. [↩]
7. ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Heise C, Sampson-Johannes A, Williams A, McCormick F, Von Hoff DD, Kirn DH. Nat Med. 1997 Jun;3(6):639-45.
   and
   An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Bischoff JR, Kirn DH, Williams A, Heise C, Horn S, Muna M, Ng L, Nye JA, Sampson-Johannes A, Fattaey A, McCormick F. Science. 1996 Oct 18;274(5286):373-6. [↩]
8. And I have no idea which, if any, is the most promising.[↩]
9. Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. David F. Stojdl, Brian Lichty, Shane Knowles, Ricardo Marius, Harold Atkins, Nahum Sonenberg & John C. Bell. Nature Medicine 6, 821 - 825 (2000) http://dx.doi.org/10.1038/77558 doi:10.1038/77558[↩]
10. One other point is that you can probably get away with a virus that isn’t completely restricted to tumor cells, because these are usually viruses that cause very mild disease anyway, so even if they can spread to normal cells it’s no more worry than exposure to a standard subway car. Maybe more concern for immunosuppressed cancer patients, of course, but likely not an insurmountable worry.[↩]