Mystery Rays from Outer Space

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

December 8th, 2010

Do TRegs discriminate?

As I’ve noted several times before, regulatory T cells are important reasons for the poor immune response to tumors. TRegs are normal components of an immune response, “designed” to keep inflammation from running riot in general and to prevent responses to self-antigens in particular. Whether it’s because tumors are mostly (though not solely) self antigens, because tumors are chronic sources of stimulation that could lead to inflammation running riot, or because tumors “learn” how to specifically trigger TReg-like responses, TRegs are common features of tumors.

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

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

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

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

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

  1. James, E., Yeh, A., King, C., Korangy, F., Bailey, I., Boulanger, D., Van den Eynde, B., Murray, N., & Elliott, T. (2010). Differential Suppression of Tumor-Specific CD8+ T Cells by Regulatory T Cells The Journal of Immunology, 185 (9), 5048-5055 DOI: 10.4049/jimmunol.1000134[][]
November 4th, 2009

Tumor TRegs are more focused than I expected

TRegs infiltrate a tumor
TRegs infiltrate into a tumor

One of the reasons the immune system doesn’t destroy tumors is the presence of regulatory T cells (TRegs) that actively shut down the anti-tumor response.  For once, there’s a little bit of encouraging news on that front.

TRegs are normal parts of the immune system.  They actively prevent other T cells (and so on) from attacking their target. 1  What’s more, TRegs are antigen-specific.  That is, they recognize a specific target, just as do other T cells, but instead of responding by, say, destroying the cells (like  cytotoxic T lymphocyte) or by releasing interferon (like a T helper cell) a TReg’s response to antigen is to prevent other T cells from doing anything in response to that antigen.  In other words, TRegs cause an antigen-specific inhibition of the conventional immune response. 2

Back to tumors.  We know that immune responses don’t routinely eliminate tumors by the time they’re detectable.  There is some evidence that lots of very small, proto-tumors, are in fact destroyed by the immune system very early on, before they’re clinically detectable, but those tumors that survive that attack seem to be pretty resistant to immune control.  At least part of that resistance is because TRegs get co-opted into the tumor’s control (see here, and references therein, for more on that).

So if TRegs are antigen-specific, and TRegs control immune responses to the tumor, what are the tumor antigens that are driving the TRegs?

I would have assumed that TRegs are looking at many, many tumor antigens, including both normal self antigens3 as well as classical tumor antigens.4  But a recent paper5 suggests, to my surprise, that this assumption is wrong.  Instead, “Tregs in tumor patients were highly specific for a distinct set of only a few tumor antigens“. 5 What’s more, eliminating TRegs cranked up the functional immune response, but only to those antigens TRegs recognized — as you’d expect, if the suppression is indeed antigen specific.

This is interesting for several reasons.  If TRegs can be specific for tumor antigens, then at least in theory ((In practice, we don’t quite have the tools yet, I think) it should be possible to turn off these TRegs while leaving the bulk of TRegs intact (and therefore not precipitating violent autoimmunity).  It also suggests that if the TRegs are only suppressing a subset of effector T cells, there’s something else preventing most effector T cells from, well, effecting.  Maybe those are antigen non-specific TRegs, or maybe there’s something else we need to know about.

I’d like to see this sort of study replicated, and a little more fine-tuning on identifying the TReg’s targets (the readout was intentionally fairly coarse here, in order to identify as many as possible).  Still, it’s an unexpected, and potentially very useful, observation.

  1. It’s still not quite clear how they do this[]
  2. There are also antigen-nonspecific TRegs, but we will ignore them for now.  They’re not as effective as the antigen-specific sort, anyway.[]
  3. Because TRegs, unlike most immune cells, can be stimulated by normal self antigens[]
  4. That is, antigens that are mutated, or dysregulated, and that therefore act as standard targets for immune cells[]
  5. Bonertz, A., Weitz, J., Pietsch, D., Rahbari, N., Schlude, C., Ge, Y., Juenger, S., Vlodavsky, I., Khazaie, K., Jaeger, D., Reissfelder, C., Antolovic, D., Aigner, M., Koch, M., & Beckhove, P. (2009). Antigen-specific Tregs control T cell responses against a limited repertoire of tumor antigens in patients with colorectal carcinoma Journal of Clinical Investigation DOI: 10.1172/JCI39608[][]
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[]
March 17th, 2008

Controlled TReg production

Saints Cosmas and Damian performing a miraculous cure by transplantation of a leg/The Master of Los Balbases.I’ve previously posted on regulatory T cells (TRegs) and their potential role in transplants. Briefly, TRegs are capable of specifically shutting off immune responses to particular antigens; they’re normal components of an immune system. TRegs can be damaging in some contexts — for example, in cancer, where it seems that TRegs often shut off immune responses to tumors, so that the tumor can escape immune clearance; and they can be beneficial in other context — for example, in some persistent virus infections, where a chronic immune response would be damaging, TRegs apparently modulate the immune response so that the virus persists but doesn’t cause severe damage.

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

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

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

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

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

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

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

  1. Verginis, P., McLaughlin, K.A., Wucherpfennig, K.W., von Boehmer, H., Apostolou, I. (2008). Induction of antigen-specific regulatory T cells in wild-type mice: Visualization and targets of suppression. Proceedings of the National Academy of Sciences, 105(9), 3479-3484. DOI: 10.1073/pnas.0800149105[][]
  2. Kretschmer, K., Apostolou, I., Hawiger, D., Khazaie, K., Nussenzweig, M. C., and von Boehmer, H. (2005). Inducing and expanding regulatory T cell populations by foreign antigen. Nat Immunol 6, 1219-1227.
    Apostolou, I., and von Boehmer, H. (2004). In vivo instruction of suppressor commitment in naive T cells. J Exp Med 199, 1401-1408.[]
January 27th, 2008

TRegs and transplants

Embryonic kidneyLast week I talked about regulatory T cells (TRegs) in cancer. TRegs are often abundant in tumors, and have been linked to poor outcome, presumably because they prevent immune rejection of the tumor. The obvious flip side of this would be in a situation where you want to prevent immune rejection — in organ transplants. TRegs have been frustratingly hard to harness for this, though. (“Tolerance is the future of transplantation, and always will be.” –Norman Shumway)

A paper in the New England Journal of Medicine1 describes an encouraging step forward in this, achieving something that is at least close to the holy grail of transplantation — organ transplants that are maintained indefinitely without immunosuppression. Normally, organ transplants are rejected by the immune system, unless they’re from an identical twin (in which case the donor organ is perceived to be “self” by the immune system). By suppressing immunity, organ transplants can “take” without rejection; usually the immunosuppression is fairly harsh at first, but can be eased up over time, suggesting that a certain degree of tolerance is reached. (Also, the donor organ probably becomes less immunogenic over time, as some of the most immunogenic cells move out of the graft or die off, leaving less immunogenic tissues behind.)

Even though today’s immunosuppression is relatively gentle and focused, it’s only gentle relative to previous brutal treatments; it still leaves the recipient susceptible to infection, so there’s always a juggling act, balancing risk of rejection with risk of infection. The goal, then, has long been to find techniques that will allow the recipient’s immune system to become tolerant of the donor organ, as is seen in tumors.

Embryonic kidneyThe paper describes five kidney transplants that were preceded by bone marrow transfer from the donor. In four of the five cases, they were able to withdraw immunosuppression altogether, and the transplant wasn’t rejected (for at least one to five years, and counting). This is particularly exciting because these transplants weren’t from HLA-matched donors, meaning they were fairly immunogenic. (The same group, and another paper in the same issue of New England Journal, have done the same thing with HLA-matched transplants,2 which is still pretty interesting; but partially-mismatched transplants are much more common these days. )

One particularly interesting observation is that the bone marrow transfer only led to temporary chimerism (i.e. the donor bone marrow didn’t take permanently, and after a while only the original recipient bone marrow cells were present); but the tolerance persisted. They were able to find lots of TRegs infiltrating the donor kidneys, though, and so they believe that the long-term tolerance is probably because of TRegs (peripheral tolerance) although in the early stages thymic effects (central tolerance) may have been more important.

Blogging on Peer-Reviewed ResearchThe same issue of New England Journal describes the case of a young liver transplant recipient who apparently had her bone marrow seeded with stem cells from the donor liver, resulting in a switch of blood type and immune system to the donor’s and, again, a complete take of the graft without immunosuppression.3 That’s the case that’s getting all kinds of press right now, but while it may turn out to be an important guide to future treatment, it was essentially pure luck — the other cases here were the result of deliberate planning and defined conditions, which means that they can be repeated; the flashy case can’t, yet.

  1. Kawai, T. et al., 2008. HLA-Mismatched Renal Transplantation without Maintenance Immunosuppression. N Engl J Med, 358(4), p.353-361. []
  2. Bühler, L.H. et al., 2002. Induction of kidney allograft tolerance after transient lymphohematopoietic chimerism in patients with multiple myeloma and end-stage renal disease. Transplantation, 74(10), p.1405-9.
    Fudaba, Y. et al., 2006. Myeloma responses and tolerance following combined kidney and nonmyeloablative marrow transplantation: in vivo and in vitro analyses. American journal of transplantation, 6(9), p.2121-33.
    Spitzer, T.R. et al., 1999. Combined histocompatibility leukocyte antigen-matched donor bone marrow and renal transplantation for multiple myeloma with end stage renal disease: the induction of allograft tolerance through mixed lymphohematopoietic chimerism. Transplantation, 68(4), p.480-4.
    Scandling, J.D. et al., 2008.
    Tolerance and Chimerism after Renal and Hematopoietic-Cell Transplantation. N Engl J Med, 358(4), p.362-368. []
  3. Alexander, S.I. et al., 2008. Chimerism and Tolerance in a Recipient of a Deceased-Donor Liver Transplant. N Engl J Med, 358(4), p.369-374. []
January 20th, 2008

TRegs and cancer

 Clark & Kupper: Blood, 1 January 2007, Vol. 109, No. 1, pp. 194-202. 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.

TReg (J Clin Invest cover)(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
• … 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.
    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[]
November 3rd, 2010

Shield or target? A downside of immune evasion

T cells & herpes simplex
T cells (green) and herpesvirus-infected cells (red)
(from Akiko Iwasaki)

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

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

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

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

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

How Not to be Seen

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

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

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

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

  1. If any[]
  2. Lampen, M., Verweij, M., Querido, B., van der Burg, S., Wiertz, E., & van Hall, T. (2010). CD8+ T Cell Responses against TAP-Inhibited Cells Are Readily Detected in the Human Population The Journal of Immunology DOI: 10.4049/jimmunol.1001774[][]
  3. As has been previously shown in mice[]
October 28th, 2010

Immunological standoff

TRegs infiltrate a tumor
TRegs infiltrate into a tumor

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

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

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

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

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

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

Reservoir Dogs StandoffA recent paper4 suggests it’s the latter:

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

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

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

  1. I’m not quite comfortable with the phrasing here, but I can’t come up with a non-lawyerly, succinct way to phrase it. TRegs are part of the immune system, and so when they’re active the immune system isn’t blocked, it’s highly functional. What’s being blocked is what we traditionally think of as an immune response — the aggressive response that causes inflammation and that kills targets — while the TReg form is the branch of the immune response that prevents all those things. When TRegs are dominant, the immune response isn’t easily visible, but it’s still an active immune response.[]
  2. Again, not happy with the term; if anyone has a more felicitious phrase, let me know[]
  3. My qualifier here is “For now”, because this is a rapidly-changing field that has kind of outstripped my ability to follow it right now; I’m not quite sure whether this is the consensus view any more[]
  4. Kuczma, M., Kopij, M., Pawlikowska, I., Wang, C., Rempala, G., & Kraj, P. (2010). Intratumoral Convergence of the TCR Repertoires of Effector and Foxp3+ CD4+ T cells PLoS ONE, 5 (10) DOI: 10.1371/journal.pone.0013623[][][]
  5. Having typed that a dozen times here, I like it less than ever[]
October 19th, 2009

Brainwashed killers

TRegs in normal skin
TRegs in normal skin

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.

Brainwashed killerIt 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[]