Mystery Rays from Outer Space

Meddling with things mankind is not meant to understand. Also, pictures of my kids

June 24th, 2009

Humans as models of human disease

You can go to the most prestigious medical center in the world and ask “How is my immune system?” and, after a short period of eye rolling and looks of amused incomprehension, you might (if they don’t just throw you out) be offered a white blood cell count (which you should probably decline). … How did we arrive at this state of affairs? A good case can be made that the mouse has been so successful at uncovering basic immunologic mechanisms that now many immunologists rely on it to answer every question. … Well, except that mice are lousy models for clinical studies. This is readily apparent in autoimmunity (von Herrath and Nepom, 2005) and in cancer immunotherapy (Ostrand-Rosenberg, 2004), where of dozens (if not hundreds) of protocols that work well in mice, very few have been successful in humans. 1

CTL attacking a tumor cell
Cytotoxic T lymphocyte attacking a tumor cell

The above (emphasis added) is from a recent manifesto from immunology giant Mark Davis.1 (He isn’t the first to make this point, of course, but when Mark Davis speaks, immunologists listen.)  Davis’s suggested solution was, among other things, to start using humans as their own models, taking advantage of the large numbers of humans who are routinely screened and overcoming the lack of experimental control by taking a “systems” approach, including large-scale data collection from healthy and ill people and large-scale informatics as part of the analysis.2 (He also comments on the “humanized” mouse approach that I mentioned briefly the other day.)

Here’s an example of the power of human models, though it’s not exactly what Davis is describing.3  I’ve mentioned before the evidence that cancers in mice are controlled by the immune system (for example, here and links therein).  In those experiments, mutant mice, lacking one or more components of the immune response, were shown to be predisposed to cancer.  There are also a couple of human studies that indicate the same thing; people on long-term immunosuppression (as in transplant recipients) are somewhat more likely to get certain kinds of cancer, for example.

CTL attacking a tumor cell
CTL attacking a tumor cell

One advantage of using humans as models of their own diseases is that there are an amazing number of well-documented mutations and disease-associated genes in humans.  Human disease is taken very seriously, it’s well funded (at least in comparison to, say, dog and cat disease); even diseases that are very rare can be identified in humans and the gene variant identified.  One such disease is Type II familial hemophagocytic lymphohistiocytosis (FHL), a rare, rapidly fatal disease caused by mutations in the perforin gene. 4  Perforin is important in cytotoxic T lymphocyte function, and it’s one of the immune genes that has been shown to be important in preventing cancers in mice.5

Are patients with Type II FHL at risk of developing cancer, like mice with targeted perforin mutations?  It’s not an easy question to ask, because most patients die relatively young.  But because these are humans, this very rare disease is nevertheless well documented, and the authors were able to search the literature for a suitable subset of patients:

… we identified a subgroup of individuals from nonconsanguineous families who possessed 2 mutated PRF1 alleles but whose onset of FHL was markedly delayed (the age at onset of 10 years or older) or even abolished. A total of only 23 such cases could be identified in the entire literature …  Ten of the individuals (Patients 14–23 inTable 1) developed manifestations of FHL without any other significant infectious or neoplastic sequelae reported. … Remarkably, in 11 of these 13 individuals (or 48% of the entire cohort of 23), the primary clinical presentation was with either B or T cell lymphoma or acute or chronic leukemia of lymphoid origin. … The very high frequency of hematological cancers in this 23-patient cohort …  is vastly in excess of that in the general population.3

There’s a lot of other interesting stuff in the paper, but this is enough to make the point: Just as in mice, perforin in humans (and therefore, the immune system) is important in preventing cancer.

I’ll leave with this now-familiar observation from the paper:

It is clearly problematic to extrapolate experimental data from inbred mouse strains to an outbred human setting where such evidence is far more difficult to gather.3


  1. DAVIS, M. (2008). A Prescription for Human Immunology Immunity, 29 (6), 835-838 DOI: 10.1016/j.immuni.2008.12.003[][]
  2. Again, of course, Davis isn’t the first to advocate this approach.[]
  3. Chia, J., Yeo, K., Whisstock, J., Dunstone, M., Trapani, J., & Voskoboinik, I. (2009). Temperature sensitivity of human perforin mutants unmasks subtotal loss of cytotoxicity, delayed FHL, and a predisposition to cancer Proceedings of the National Academy of Sciences, 106 (24), 9809-9814 DOI: 10.1073/pnas.0903815106[][][]
  4. Familial hemophagocytic lymphohistiocytosis. Primary hemophagocytic lymphohistiocytosis.
    Henter JI, Aricò M, Elinder G, Imashuku S, Janka G.
    Hematol Oncol Clin North Am. 1998 Apr;12(2):417-33[]
  5. Perforin-mediated Cytotoxicity Is Critical for Surveillance of Spontaneous Lymphoma.
    Mark J. Smyth, Kevin Y.T. Thia, Shayna E.A. Street, Duncan MacGregor, Dale I. Godfrey, and Joseph A. Trapani.
    The Journal of Experimental Medicine, Volume 192, Number 5, September 5, 2000 755-760[]
May 26th, 2009

Why (some) similar tumors are similar

Caco2 colon carcinoma cells
Caco2 colon carcinoma cells

One of my long-standing questions now has at least a partial answer, or maybe a pathway toward a partial answer.

My question was, “Why are different tumors the same?” That is, why do tumors of the same type often seem to have similar immunological changes?

Viruses of the same kind (all herpes simplex viruses, say) all avoid immunity in the same way because they all have a common ancestor from which they inherited their immune evasion molecules. But tumors have no common ancestor; each tumor has to grope around and find its own solution to common problems independently. So why should tumors of a particular tissue converge on common solutions? What would make a certain pathway a simple solution for colon carcinomas, but not for bladder tumors? And yet, apparently that’s what happens. Even though many different tumor types become non-immunogenic, tumors of a particular tissue type often reach non-immunogenicity by a similar path. For example:

… distinct molecular events underlie HLA class I loss, depending on the aetiology of the tumours; Lynch syndrome-related cancers presented with mutations in the β2-m molecule, while sporadic microsatellite-unstable tumours mainly showed alterations in the antigen-processing machinery components 1

When I posed this question a couple of months ago, I didn’t have an answer. I said

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

EH, in the comments, suggested a couple more possibilities: Perhaps destruction of a particular pathway is “a nice side benefit of destroying some yet undiscovered tumor suppressor important for melanoma or colon cancer? Or maybe the loss of certain repair genes common to a cancer type (like BRCA) leads to selective chromosomal instability in parts of chromosome 6?

It turns out, unsurprisingly, that I’m not the only one to ask the question. There is no general answer, but apparently a partial answer has been kicking around for a while now, and Hans Morreau’s group is starting to put some of the pieces together.2 As their model, Morreau’s group looked at MUTYH-associated polyposis (“MAP”)-associated tumors. (MAP is a heritable defect in DNA repair, so MAP patients have high rates of mutagenesis and usually develop colon carcinomas.) They reasoned that

MAP tumours could be more prone to stimulate a cytotoxic T-cell-mediated immune response, due to their frequent generation of aberrant peptides. Hence, these tumours could also be subjected to a strong selective pressure favouring the outgrowth of cancer cells that acquire an immune evasive phenotype.

DNA Repair
DNA Repair

In other words, the argument is that the group of tumors that lose (or reduce) DNA repair are much more likely to throw out mutant proteins. These mutant proteins are targets for the immune response (because they’re no longer “self” antigens), so to survive the immune attack the tumor has a strong selection for loss of immunogenicity (and also has the high mutation rate that allows them to rapidly mutate away from immunogenicity).

Sure enough, the tumors did frequently (72%) have defects in antigen presentation. (In fact, because of the way they measured HLA expression — by immunohistochemistry rather than sequencing — I would bet that the rate of functional defects was actually much higher than that.) They conclude that this “provides additional evidence that tumours carrying defects in DNA base repair mechanisms are more prone to undergo immune escape mechanisms.2  Since they don’t formally compare to other tumor types, I don’t think they can really say “more prone” — you’d have to use the same techniques to look at tumors from non-MAP patients to be able to say that. Still, I do think that is a significantly higher rate than has been turned up in previous studies using similar techniques,3 so I’ll tentatively accept that conclusion.

This doesn’t really explain why similar tumors target similar components, but it’s at least a conceptual connection between different tumor types and an underlying pathway. I’d be interested in a more large-scale screen, looking at cancers with stronger and weaker mutator phenotypes to see if common pathways emerge. One may already have popped up, since the authors note here that expression of β2-m was frequently lost4 in several tumors with DNA repair defects:

Although speculative, it is interesting to underline that carcinomas derived from both MAP and Lynch syndromes preferentially lose β2-m expression coupled to HLA class I deficiencies. A functional explanation for these observations remains elusive, but perhaps distinct reactions (both qualitative and quantitative) by the immune system, depending on the age of onset of the tumours, could condition the type of mechanisms that lead to HLA class I expression deficiencies. 2

Again, we would need to compare to other tumor types to see if this really is more frequent, but overall it feels as if there’s a hint of a pathway here. At least there are some specific questions that can be asked.


  1. de Miranda, N., Nielsen, M., Pereira, D., van Puijenbroek, M., Vasen, H., Hes, F., van Wezel, T., & Morreau, H. (2009). MUTYH-associated polyposis carcinomas frequently lose HLA class I expression-a common event amongst DNA-repair-deficient colorectal cancers The Journal of Pathology DOI: 10.1002/path.2569
    Referencing
    Dierssen JWF, de Miranda NFCC, Ferrone S, van Puijenbroek M, Cornelisse CJ, Fleuren GJ, et al. HNPCC versus sporadic microsatellite-unstable colon cancers follow different routes toward loss of HLA class I expression. BMC Cancer 2007; 7: 33[]
  2. de Miranda, N., Nielsen, M., Pereira, D., van Puijenbroek, M., Vasen, H., Hes, F., van Wezel, T., & Morreau, H. (2009). MUTYH-associated polyposis carcinomas frequently lose HLA class I expression-a common event amongst DNA-repair-deficient colorectal cancers The Journal of Pathology DOI: 10.1002/path.2569[][][]
  3. Other studies, especially those of Soldano Ferrone, have turned up much higher rates of functional HLA class I defects, but they’ve used more focused, and more difficult and expensive, techniques to reach that conclusion.[]
  4. β2-m is a physical component of the MHC class I complex[]
April 9th, 2009

Why are different tumors the same?

Hierarchical clustering of breast carcinomas, Turashvili et al 2007
Hierarchical clustering of breast carcinomas1

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

What I mean is this:

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

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

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

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

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

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

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


  1. Turashvili et al. BMC Cancer 2007 7:55   doi:10.1186/1471-2407-7-55[]
  2. Seliger, B. (2008). Molecular mechanisms of MHC class I abnormalities and APM components in human tumors Cancer Immunology, Immunotherapy, 57 (11), 1719-1726 DOI: 10.1007/s00262-008-0515-4[][]
  3. barring such weird things as canine transmissible venereal tumor and Tasmanian Devil facial tumors; see here for more on those[]
  4. The comparison is, of course, viruses.  A herpesvirus of chickens, and one of humans, may both use immune evasion mechanisms, but they have a common ancestor even if it’s a couple of hundred million years ago.[]
April 6th, 2009

Inflammation and cancer: Proof that the universe hates us?

Metchnikov - Lecons sur la pathologie
Metchnikov: “Lecons sur la pathologie” (1892)

There are times when you just feel like the universe is out to get you. For example, we know that inflammation can drive tumor formation; but a paper just came out that suggests reducing inflammation can also drive tumor formation. 1 It doesn’t seem fair.

I’ve previously mentioned the link between inflammation and tumorigenesis, which is probably at least partly because the inflammation produces reactive oxygen and nitrogen species (RONS ) that are tumorigenic.

I’ve also talked about the link between reduced inflammation and ongoing tumors (for example, here, here, and here). What seems to be going on here is that regulatory T cells (TRegs) are induced by tumors, and these TRegs shut down anti-tumor immunity.

So far, these findings aren’t really contradictory. Increasing inflammation before a tumor is present makes tumors more likely to form. After the tumor has formed, reducing inflammation makes the tumor more likely to persist. The universe-is-against-us part comes from the suggestion that reducing inflammation (via TRegs) before tumor formation, also makes the tumors more likely to form.

This may be a special case. The paper from Philip Dennis’s group 1 looked at a specific set of cancers, those associated with K-Ras mutations (linked to smoking-induced lung cancer). K-Ras activation itself triggers inflammation (for reasons I, at any rate, don’t understand). When K-Ras is activated, as well as inflammation, TRegs move into the area, and presumably reduce the inflammation. Depleting the TRegs (and therefore increasing the inflammation) decreased the number of tumors by 75% — the opposite of what you’d expect if inflammatory RONS were driving tumorigenesis.

Smoking / cancer

A common feature linking smoking induced K-Ras mutations in human lung cancer and preclinical models driven by tobacco carcinogens that cause K-Ras mutations is inflammation. In both cases, the presence of Foxp3+ cells is likely important for limiting the extent of inflammation and tissue damage, albeit at a potential cost of promoting tumorigenesis. 1

In later-stage tumors the situation became more consistent with other work — getting rid of TRegs reduced the tumors, suggesting that these tumors were depending on TRegs to prevent immune clearance:

Aggressive and invasive K-Ras-induced adenocarcinomas (IO33 and K-RasLA2) remained sensitive to more direct targeting of Foxp3+ cells through a neutralizing anti-CD25 antibody or genetic deletion. This indicates that direct Treg cell depletion strategies that are being evaluated clinically could have therapeutic value in more advanced stages of K-Ras driven lung cancer. 1

My question here is whether the early inflammation is kind of a red herring. Could the TReg depletion in the early stages be reducing the anti-tumor immune response in a specific way, just as in the later stages of tumor formation? That is, could the TReg depletion lead to a tumor-specific immune response, which prevents tumors from forming? In this case the inflammation could still be driving the tumor formation, but the increase in tumor formation would be outweighed by the simultaneous increase in anti-tumor immunity. I don’t know quite how to test this, but perhaps doing the same experiment in mice lacking, say, CD8 T cells might be interesting. (Such mice should still have the early inflammation and the TRegs, but may have a less effective immune response. It’s not a perfect experiment, though, for reasons that are probably too complex to go into here.)


  1. Granville, C., Memmott, R., Balogh, A., Mariotti, J., Kawabata, S., Han, W., LoPiccolo, J., Foley, J., Liewehr, D., Steinberg, S., Fowler, D., Hollander, M., & Dennis, P. (2009). A Central Role for Foxp3+ Regulatory T Cells in K-Ras-Driven Lung Tumorigenesis PLoS ONE, 4 (3) DOI: 10.1371/journal.pone.0005061[][][][]
February 24th, 2009

More chemotherapy and tumor immunity

U-118 glioma cells (Nikon Microscopy Gallery)
U-118 Glioma cells 

In one of the earliest posts I made to Mystery Rays,  I commented on an exciting anti-tumor finding. 1  Basically, the suggestion was that when chemotherapy of tumors works, it doesn’t actually work by killing all the tumor cells; or at least not directly.  Instead, the authors said, chemo works because the dying tumor cells release adjuvants (immune stimulants),2 and what actually eliminates the tumor is the specific immune response to the tumor, driven and enhanced by the immune stimulants.   In other words, the chemotherapy is actually acting like the adjuvant in an anti-cancer vaccine. 

Presumably the reason this form of adjuvant is effective, whereas just giving an adjuvant to a cancer patient is not, is that it’s so tightly linked to the tumor you want the immune system to target.   

In the subsequent year and a half there have been a handful of papers looking more at this phenomenon (not counting the review papers, which have churned out at a great rate). For example, it’s been shown that dying tumor cells are cross-presented efficiently due to HMGB1 and similar immune stimulators.3    Now, there’s a more specific support of the concept. 4 

I don’t know if the Apetoh et al paper was the initial impetus for the latest one, or (more likely) if it was already in progress, but a any rate, unless I’ve missed one, this is the second study that directly looks at HMGB1 effects on tumor regression following tumor cell death, and it reaches basically the same conclusion as the first paper.  This was strictly a mouse study, but they used a highly aggressive brain tumor (glioblastoma multiforme).  As well as chemotherapy they used a gene therapy approach, thus managing to hit four of my Mystery Rays buzzwords (tumor immunity, immunity to viruses, innate adjuvants, and if you’re generous oncolytic viruses as well) all at once.  (What would a Mystery Rays Bingo Card look like?)

Glioblastoma
Glioblastoma

If the model is correct, then what you really want to do when treating tumors is a double whammy — kill tumor cells, and simultaneously attract in immune cells.  Curtin et al did this by injecting two recombinant adenoviruses directly into the tumor; one rendered the infected cells sensitive to chemotherapy, and the other attracted dendritic cells (DC are important cells for initiating immune responses). Following chemo, the tumors regressed dramatically (about half the mice survived, compared to 100% death without treatment).  And both of the viruses were necessary; without the immune system, killing the cells wasn’t enough, and without tumor cell death, the immune system couldn’t do enough. 

The same treatment in mice without TLR2 did nothing.  TLR2 is an innate immune recognition molecule that triggers inflammation in the presence of (among other things) HMGB1, which is released from dying cells.  This is similar to the Apetoh et al paper, and Curtin et al took the studies a little further by showing that TLR2 had to be on dendritic cells (as opposed to the tumor itself). What’s more, when all the parts were in place (tumor cell death, DC  present, DC expressing TLR2) there was a strong specific anti-tumor cytotoxic T lymphocyte response, while without the TLR2 stimulation there was little such response.

In other words, they’ve firmed up the probable pathway by which chemotherapy eliminated these tumors. The chemotherapy killed cells directly, and the dying cells released HMGB1; the HMGB1 activated dendritic cells in the tumor, by triggering their TLR2 receptor; the activated DC then in turn activated CTL specific for the tumor; and the CTL then completely eliminated the tumor and prevented the remains from regrowing.  (Some of the steps in this pathway remain formally unproven, but the data are consistent with this model.) 

It’s not clear, yet, if this is the universal explanation for successful chemotherapy, or whether chemo sometimes or often works by killing the tumor directly with no requirement for immunity. The authors here looked at several different tumors, expanding on the more correlative data from Apetoh et al., and so far all the cancers that have been checked fall into the immune clearance category.  So, while it’s a long, long way from the bedside, it’s certainly encouraging. 

In conclusion, the results reported provide compelling evidence for the role played by HMGB1 in mediating the efficacy of antiglioma therapeutic regimes that are based on tumor cell killing strategies … [C]ancer immunotherapies coupled with effective cell killing modalities may be necessary to achieve therapeutically relevant antitumor efficacy. 4


  1. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Apetoh et al. Nature Medicine 13, 1050 – 1059 (2007)  doi:10.1038/nm1622

    Also this review:

    Apetoh, L., F. Ghiringhelli, A. Tesniere, A. Criollo, C. Ortiz, R. Lidereau, C. Mariette, N. Chaput, J. P. Mira, S. Delaloge, F. Andre, T. Tursz, G. Kroemer, and L. Zitvogel. 2007. The interaction between HMGB1 and TLR4 dictates the outcome of anticancer chemotherapy and radiotherapy. Immunol. Rev. 220:47-59.

    and this one:

    Apetoh, L., A. Tesniere, F. Ghiringhelli, G. Kroemer, and L. Zitvogel. 2008. Molecular interactions between dying tumor cells and the innate immune system determine the efficacy of conventional anticancer therapies. Cancer Res. 68:4026-4030.

    and this review from a different group:

    Campana, L., L. Bosurgi, and P. Rovere-Querini. 2008. HMGB1: a two-headed signal regulating tumor progression and immunity. Curr. Opin. Immunol. 20:518-523.[]

  2. Specifically, the dying cells release a protein called HMGB1, which has been shown to be an endogenous adjuvant[]
  3. Brusa, D., S. Garetto, G. Chiorino, M. Scatolini, E. Migliore, G. Camussi, and L. Matera. 2008. Post-apoptotic tumors are more palatable to dendritic cells and enhance their antigen cross-presentation activity. Vaccine 26:6422-6432.[]
  4. James F. Curtin, Naiyou Liu, Marianela Candolfi, Weidong Xiong, Hikmat Assi, Kader Yagiz, Matthew R. Edwards, Kathrin S. Michelsen, Kurt M. Kroeger, Chunyan Liu, A. K. M. Ghulam Muhammad, Mary C. Clark, Moshe Arditi, Begonya Comin-Anduix, Antoni Ribas, Pedro R. Lowenstein, Maria G. Castro (2009). HMGB1 Mediates Endogenous TLR2 Activation and Brain Tumor Regression PLoS Medicine, 6 (1) DOI: 10.1371/journal.pmed.1000010[][]
February 3rd, 2009

Virus vs tumor (vs ninja vs pirate): Computational oncolysis

Jennerex oncolytic virus
Jennerex” virus (green) replicating within
a tumor mass.

Viruses infect cells, and quite often (depending on the type of virus) destroy the cell they’re infecting. Usually having your cells destroyed is a bad thing for the host, because you need those cells. But there are some cells that you don’t want to have, and it might be convenient to have a controlled virus destroy those cells for you.

The obvious example would be cancer cells, and in fact there’s a flourishing research industry that is trying to harness the destructive power of viruses to eliminate tumors. The trick, of course, is to have the virus infect only the cancer cells, not the normal healthy cells you want to keep; and the general approach is to take advantage of some of the common features of viruses and cancers. Cancers have to mutate to overcome some of the same cellular controls that viruses do (both viruses and cancers need to overcome the regulation of uncontrolled genome replication, for example). If you cripple some of these viruses, then, they can’t replicate in normal cells, but can replicate in, and destroy, cancer cells that have mutated the appropriate pathway.

(I talk about oncolytic viruses in more detail here.)

As often happens with these intriguing cancer treatments, oncolytic viruses seem to work sometimes, and not to work sometimes, and it’s not always clear why not. In a paper in PLoS One the other day,1 Wodarz and Komarova attempt to come up with a way of predicting which tumors will and will not respond to oncolytic virus therapy, using a computational approach.

As I think I’ve said before, I’m excited by the concept of computational biology. (I’m not so much talking about bioinformatics as such here, but rather about attempts to model AND PREDICT complex biological processes.) But I’ve been kind of disappointed by some of the process. It’s seemed to me that when simple processes are modeled we haven’t really learned very much new, and when complex processes are modeled the assumptions are often too simplistic to make a reasonable prediction. I don’t think we’re at a point where we can usefully model an immune system, for example.

Wodarz oncolysis model
Growth of cancer in a mouse in the presence of  oncolytic virus 1

However, I do think there are a class of problems in the middle where the approach is more successful, and though I’m not really able to critically assess their results I think over the years Dominik Wodarz has done a good job of identifying these problems. Questions like the emergence of drug resistance in cancer,2 effects on vaccination on HIV,3 and so on seem like the kind of problem where mathematical analysis can actually get a handle on the issues and help guide research to some extent. Again, I don’t feel that I can really judge the results, but I like the approach. What’s more, Wodarz seems to at least consider experimental evidence in his analyses, which isn’t always the case in these computational things.

(I don’t think it’s a coincidence that many of the questions he’s asked are microcosmic versions of ecological issues.  My impression is that population biology has a much longer and more successful history of mathematical analysis than do cell and molecular biology.)

This particular paper leads to a conclusion that, once reached, seems fairly obvious in hindsight, but it’s one I haven’t seen explicitly made before. (I am not in the field, and it may be taken for granted.  That said, one hallmark of a successful prediction is that everyone immediately says they knew it all along.) Briefly, tumor growth rate per se turn out to be relatively unimportant, and growth patterns are important. If the cancerous cells are relatively spread out in the tumor, then an oncolytic virus has a good chance of eliminating the tumor; whereas if the cancer cells are in clumps, the virus is much less effective. This is simply because in the clumpy masses most of the infected cells are contacting already-infected cells, and the only route to reach new targets is from the surface of the clump, so spread is inefficient.

In one group, virus growth is relatively fast because the infected cells are dispersed among the uninfected cells rather than being clustered together. In this case most infected cells contribute to virus spread. In these models, there is a clear viral replication rate threshold beyond which the number of cancer cells drops to levels of the order of one or less, corresponding to extinction in practical terms. … In the other category, infected cells are assumed to be clustered together to some degree in a mass, which might be realistic for solid tumors. In this case, only the infected cells located at the surface of the cluster contribute to virus spread because they are in the vicinity of uninfected cells. … In this scenario, virus therapy is more difficult. 1


  1. Dominik Wodarz, Natalia Komarova (2009). Towards Predictive Computational Models of Oncolytic Virus Therapy: Basis for Experimental Validation and Model Selection PLoS ONE, 4 (1) DOI: 10.1371/journal.pone.0004271[][][]
  2. Drug resistance in cancer: principles of emergence and prevention. Komarova NL, Wodarz D. Proc Natl Acad Sci U S A. 2005 Jul 5;102(27):9714-9.[]
  3. Immunity and protection by live attenuated HIV/SIV vaccines. Wodarz D. Virology. 2008 Sep 1;378(2):299-305.[]
December 7th, 2008

How do TRegs work?

TReg (Artist's impression from BioLegend)After the “suppressor T cell” debacle of the 1980s, there was an embarrassed pause for a few years before people dipped their toes back into the suppressor T cell water; but the underlying phenomenon itself is a very strong and important one, and by the late 1990s and early 2000s researchers were again studying the cells, renaming them “regulatory T cells” (TRegs) in the process. Since the phenomenon is so strong, the field quickly exploded (from two papers mentioning TRegs in 2000, to 780 this year). We now know where TRegs are made and, mostly, how they’re made; we know what they look like and which cells they talk with; we know of various ways to make them in the lab; we know diseases where they’re overactive, and diseases where they’re underactive.  I’ve talked about these things quite a bit here.  

We didn’t know, though, how they actually work. Do they act directly on their target T cells, or via intermediaries? Do they have to contact their targets, or can they act at a distance? What molecules deliver their “regulatory” signals, and what molecules receive the signal? Well, we still don’t really know the answers to most of those questions, but a paper last month1 brought the answers a lot closer with evidence that CTLA4 is essential for TRegs to have their regulatory effect.

TRegs in skin
TRegs in normal skin

This isn’t a new idea; it was first put forward in one of the very early TReg papers, way back in 20002. The difference is that the earlier papers couldn’t cleanly distinguish TReg-specific effects of CTLA4 from its myriad other effects. CTLA4 is a very broad-acting molecule with lots of immunosuppressive (or if you prefer, immunoregulatory) activities. In the present paper, Wing et al managed to eliminate CTLA4 specifically from TReg cells, leaving its other activities intact. These TReg-specific knockouts still developed the horrible, fatal autoimmune diseases characteristic of TReg deficiencies.

So CTLA4 is essential for TReg function. This is especially interesting because there’s a lot of clinical interest in CTLA4; for example, blocking CTLA4 has been effective in generating (or regenerating) immunity to cancers, at least in experimental models. The rationale for this has been because signaling through CTLA4 on “conventional” (that is, effector, as opposed to regulatory) T cells reduces or blocks their activity;3 but now this is directly linked to TReg activity as well.

The link between TRegs, CTLA4, and tumor immunity was really emphasized in the Wing et al paper. In one experiment, they demonstrated that mice with normal TRegs were not able to reject a tumor (“All recipients of FIC splenocytes died of tumor progression within a month“), whereas mice with the knockout TRegs (that is, TRegs lacking CTLA4) were able to control it (“In contrast, recipients of CKO splenocytes halted the tumor growth, with the majority surviving the 6-week observation period, during which 60% of them completely rejected the tumor“).

Obviously, you don’t want to eliminate TReg function willy-nilly even in cancer patients; remember that these mice died of autoimmune disease when they were a couple of months old. But if there’s a way of localizing CTLA4 blockade so that the tumor-specific TRegs alone are affected, this could be very interesting.


  1. K. Wing, Y. Onishi, P. Prieto-Martin, T. Yamaguchi, M. Miyara, Z. Fehervari, T. Nomura, S. Sakaguchi (2008). CTLA-4 Control over Foxp3+ Regulatory T Cell Function Science, 322 (5899), 271-275 DOI: 10.1126/science.1160062

    Also see the commentary by Ethan Shevach:
    E. M. Shevach (2008). IMMUNOLOGY: Regulating Suppression Science, 322 (5899), 202-203 DOI: 10.1126/science.1164872[]

  2. Cytotoxic T Lymphocyte–Associated Antigen 4 Plays an Essential Role in the Function of Cd25+Cd4+ Regulatory Cells That Control Intestinal Inflammation.  S. Read, V. Malmstrom, F. Powrie, J. Exp. Med. 192, 295 (2000).[]
  3. For a review, see:
    Principles and use of anti-CTLA4 antibody in human cancer immunotherapy. Karl S Peggs, Sergio A Quezada, Alan J Korman and James P Allison Curr Opin Immunol. 2006 Apr;18(2):206-13. doi:10.1016/j.coi.2006.01.011[]
December 4th, 2008

Controlled TRegs: The future is (almost) now

TRegs (JCI)Our bodies are crammed with millions of tiny time bombs: lymphocytes that could begin to attack our own bodies, causing lethal autoimmune disease. Traditionally, it was said that these self-reactive lymphocytes were rare, because they were eliminated during their development and were never allowed to reach maturity. But it’s been known for quite a few years now that that’s not entirely true. The vast majority of self-reactive T cells may, indeed, be destroyed in the thymus, but by no means all. (Something like a couple million T cells leave a happy, functioning thymus every day. If central tolerance is 99.999% perfect, then 10 self-reactive T cells will enter the system every single day — and it only takes a couple of T cells to initiate a lethal disease.)

Why don’t we all die as infants of autoimmune attack, if circulating self-reactive T cells are so (relatively) common? As with just about everything in our body, there are redundant systems. For autoimmunity, the next line of defense is the regulatory T cell (TReg).

TRegs were identified as a phenomenon long ago, in the 1960s and 1970s; but the concept abruptly fell out of favor in 1984 (for fascinating and rather embarrassing reasons I talked about here), and it wasn’t until the new millennium that immunologists really returned to the field (first firmly changing the name from “suppressor T cells” to “TRegs” to keep their feet out of the muck), and the field really exploded 5 or 6 years ago.

TRegs have proved more important and powerful than just about anybody would have believed ten years ago. Even very powerful immune responses can be controlled by TRegs; strong TReg responses can actually allow a complete “take” of an organ transplant, for example (I mentioned some examples here).

 TRegs infiltrate tumor
Regulatory t cells infiltrate tumor tissue

As well as transplants, being able to turn on TRegs has potential for lots of other diseases. Autoimmunity, obviously, could be controlled this way; but also, less obviously, it’s possible that some virus diseases might benefit from a TReg response. HIV infection, for example, is exacerbated when T cells are activated, and monkeys with SIV are resistant to disease when their T cells are less reactive (see here and here); could a controlled TReg response reduce the harmful activation associated with HIV? It may seem counterintuitive to try to treat a viral disease by reducing immunity, but there is some precedent. Rodents infected with hantaviruses develop a TReg response and don’t have much disease (see here), while humans react with a more conventional immune response and have severe disease. And recently, it was shown that elite suppressors of HIV may have an exceptionally strong TReg response.1

Conversely, there are lots of instances where we’d like to turn off TRegs, in a controlled way. Tumors are often associated with TRegs, which very likely prevent a cleansing immune response to the tumor (discussed here). And the well-known observation that the elderly often have poor immunity against various pathogens is at least partly because TRegs build up over time.

This is a very fast-moving field, and there are a several recent papers that show exciting advances. One is a huge basic step forward, and I’ll talk about that later. The others2 are technical advances, developing new techniques (that are much less cumbersome and finicky than some of the previous approaches) to generate large numbers of TRegs in a controlled way. The obvious use for this is in transplants:

The ex vivo expansion protocol that we describe will very likely increase the success of clinical Treg-based immunotherapy, and will help to induce tolerance to selected antigens, while minimizing general immune suppression. This approach is of particular interest for recipients of HLA mismatched transplants.3

Controlled TRegs have been a holy grail of transplant biology for years, and it’s exciting to see that we may finally be entering an era when TRegs can be produced and used as tools.


  1. Preservation of FoxP3+ regulatory T cells in the peripheral blood of human immunodeficiency virus type 1-infected elite suppressors correlates with low CD4+ T-cell activation.
    Chase AJ, Yang HC, Zhang H, Blankson JN, Siliciano RF
    J Virol 2008 Sep 82(17):8307-15[]
  2. Including, but not limited to:
    W. Tu, Y.-L. Lau, J. Zheng, Y. Liu, P.-L. Chan, H. Mao, K. Dionis, P. Schneider, D. B. Lewis (2008). Efficient generation of human alloantigen-specific CD4+ regulatory T cells from naive precursors by CD40-activated B cells Blood, 112 (6), 2554-2562 DOI: 10.1182/blood-2008-04-152041

    In Vitro Expanded Human CD4+CD25+ Regulatory T Cells are Potent Suppressors of T-Cell-Mediated Xenogeneic Responses. Wu, Jingjing; Yi, Shounan; Ouyang, Li; Jimenez, Elvira; Simond, Denbigh; Wang, Wei; Wang, Yiping; Hawthorne, Wayne J.; O’Connell, Philip J. Transplantation Volume 85(12), 27 June 2008, pp 1841-1848.

    Jorieke H. Peters, Luuk B. Hilbrands, Hans J. P. M. Koenen, Irma Joosten (2008). Ex Vivo Generation of Human Alloantigen-Specific Regulatory T Cells from CD4posCD25high T Cells for Immunotherapy PLoS ONE, 3 (5) DOI: 10.1371/journal.pone.0002233

    and a review in Piotr Trzonkowski, Magdalena Szary?ska, Jolanta My?liwska, Andrzej My?liwski (2008). Ex vivo expansion of CD4+CD25+ T regulatory cells for immunosuppressive therapy
    Cytometry Part A, 9999A DOI: 10.1002/cyto.a.20659
     []

  3. Jorieke H. Peters, Luuk B. Hilbrands, Hans J. P. M. Koenen, Irma Joosten (2008). Ex Vivo Generation of Human Alloantigen-Specific Regulatory T Cells from CD4posCD25high T Cells for Immunotherapy PLoS ONE, 3 (5) DOI: 10.1371/journal.pone.0002233[]
November 21st, 2008

Watch them die

Here’s another extraordinary movie, taken from:
A. A. Cohen, N. Geva-Zatorsky, E. Eden, M. Frenkel-Morgenstern, I. Issaeva, A. Sigal, R. Milo, C. Cohen-Saidon, Y. Liron, Z. Kam, L. Cohen, T. Danon, N. Perzov, U. Alon (2008). Dynamic Proteomics of Individual Cancer Cells in Response to a Drug Science DOI: 10.1126/science.1160165

This shows cancer cells responding to a drug used in chemotherapy (camptothecin).  The drug kills many, though not all, of the cells; the paper is aimed at finding correlates between various cellular proteins, and the cells’ ability to survive.  But the move here can be interpreted more simply.  We see the cancer cells, untreated for 24 hours, rapidly dividing and squirming around.  (There are lots of clear examples of cells dividing; for example,  a little bit toward 7:00 from center, at 5 hours; and just below center, at 19 hours.). At 24 hours, the drug is added; within another 12 hours, the cells slow down, and around 48 hours we see them starting to die (dead and dying cells are helpfully boxed).  




 

In other news, I’ll be at the Autumn Immunology Conference in Chicago this weekend.  Should be good.

September 14th, 2008

Immune clearance of brain cancer

The brain (William Say, 1829)
The brain (William Say, 1829)

The other day I talked about Jim Allison’s paper1 that proposed (among other things) that irradiation of tumors might help the immune system detect them. A quite unrelated paper2 seems to have almost accidentally helped test that suggestion.

Allison’s group was looking at the puzzling fact that circulating anti-tumor T cells don’t seem to correlate well with effective anti-tumor immune responses. They suggest that part of the problem is that the blood vessels that supply tumors are often abnormal, and these altered blood vessels don’t allow T cells to enter the tissues properly. However, irradiating those blood vessels made them closer to normal, allowed the T cells to enter the tumor more effectively, and led to more complete immune clearance of the tumor.

The new paper I mentioned2 wasn’t specifically looking at tumor vasculature at all. They were working with a model of brain cancer (a particularly difficult nut to crack, immunologically). They used transgenic mice, that express a tumor antigen (the SV40 virus large T antigen) in some brain cells; these mice develop brain cancer and die by 3 or 4 months of age. They’re immunologically tolerant to the tumor antigen, as you’d expect; but transferring tumor antigen-specific T cells from non-transgenic mice helped them survive longer. In this particular paper, they try to narrow down the specific conditions that allow immune rejection of the tumor.

T cells attacking tumor cellThey found a number of important factors, but the interesting one in light of the Allison paper was that “successful adoptive immunotherapy of T Ag-induced tumors still required prior conditioning of the host with gamma irradiation”.  Just transferring the T cells helped a bit, delaying death by a couple of months; but irradiating the tumors and transfering the T cells completely eliminated the tumors. They didn’t specifically look at the tumor blood vessels (the Allison paper wasn’t out when this work was done, of course), but they did note that irradiation correlated with increased T cell accumulation in the brain.

Another interesting point is that the irradiated tumors were cleared even when only a single T cell epitope was targeted. That’s an important finding, because one might expect that the tumor might be able to recur because of immune escape. (That is, tumor cells with fortuitous mutations in that epitope would not be killed by the T cells, and would be able to regrow and recur.) In fact, that might have happened with the non-irradiated mice, in which the tumor shrank briefly then rapidly recurred. (They didn’t check the epitope, so it’s equally possible that something like tolerance or deletion of the T cells was involved in this situation.) Still, it seems that, at least in this model, you can get rid of a tumor altogether, even if you only have a single tumor epitope to work with. Given the limited number of tumor epitopes we have to work with, that’s pretty encouraging.


  1. S. A. Quezada, K. S. Peggs, T. R. Simpson, Y. Shen, D. R. Littman, J. P. Allison (2008). Limited tumor infiltration by activated T effector cells restricts the therapeutic activity of regulatory T cell depletion against established melanoma Journal of Experimental Medicine, 205 (9), 2125-2138 DOI: 10.1084/jem.20080099[]
  2. Angela M. Tatum, Lawrence M. Mylin, Susan J. Bender, Matthew A. Fischer, Beth A. Vigliotti, M. Judith Tevethia, Satvir S. Tevethia and Todd D. Schell.
    CD8+ T Cells Targeting a Single Immunodominant Epitope are Sufficient for Elimination of Established SV40 T Antigen-Induced Brain Tumors.
    The Journal of Immunology, 2008, 181: 4406-4417.
    [][]