Mystery Rays from Outer Space

This post is the short form of the now very long post here.  This is just the summary of what I’ve done and what I’m concluding; if you want to see why plus the grimy details, check there.

1. My question: The regular influenza vaccine this year included an H1N1 virus (A/Brisbane/59/2007 (H1N1)). Will that protect against infection with the new H1N1 strain?
2. My first question was, How similar is this H1N1 to that in the current vaccine? My answer:  Not very. The vaccine strain is only 79% identical to the present strain, which isn’t terrible but isn’t very good either.  For context, the H3 hemagglutinin in the vaccine is around 45% identical to the H1; we know there’s almost no cross-reactivity between these types.  The HA from the B/Florida/4/2006, a B rather than an A strain and expected to very quite different, is about 30% identical.  See the alignment and the phylogenetic tree in that post.
3. Are the differences between the viruses especially large at the places where protective antibodies bind?  If so, then we’d expect little cross-protection between the strains.
4. First, we have to figure out where protective antibodies bind.  A paper from 1982 (Caton et al., (1982). The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype) Cell, 31 (2), 417-427 DOI: 10.1016/0092-8674(82)90135-0) offered several regions of H1 where protective antibodies might bind.
5. I ran a script on a few hundred different H1 proteins from different viruses, and determined that the regions Gerhard’s group said bind to protective antibodies tend to be highly variable between viruses.  This is presumably the result of selection at that region to avoid antibody responses. So this is consistent with Gerhard’s conclusion and I’m willing to go with those regions as antibody binding sites.
6. I compared the amino acids in the regions Gerhard flagged between the vaccine strain, and the new H1N1 strain. If those regions are very similar then I would expect good cross-protection; if very different, I would expect little cross-protection.  The result (shown below) is that there are extensive differences between the putative protective antibody binding sites.

Conclusion (based on lots of assumptions!):

You can see that of the five antibody-binding sites (Sa, Sb, Ca1, Ca2, and Cb), four are really very different, while one is quite similar.  Even in that one,  the single difference, from Ser to Pro, is a drastic change that would probably significantly reduce antibody binding.  So most antibodies wouldn’t bind well to the new H1N1.  However, the most similar region (”Sa”) is the one that Gerhard flagged as the most important for antibody binding, so I’m leaning to the concept that there probably will be a little bit of cross-protection, but not a lot.

But what I would really like to see is an actual experiment, testing cross-protection and cross-reactivity between the epidemic and the vaccine H1N1.

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.

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[↩][↩][↩][↩]

It was previously thought that Drosophila is unable to spread systemically an RNAi response, based on observations that endogenously expressed RNA hairpins do not spread from cell to cell. However, we demonstrate that, upon virus infection, infected cells spread systemically a silencing signal that elicits protective RNAi-dependent immunity throughout the organism. … In striking parallel to vertebrates, flies also rely on systemic immunity, albeit in this case the virus-specific signal is dsRNA-based. ¹

1. Saleh, M., Tassetto, M., van Rij, R., Goic, B., Gausson, V., Berry, B., Jacquier, C., Antoniewski, C., & Andino, R. (2009). Antiviral immunity in Drosophila requires systemic RNA interference spread Nature, 458 (7236), 346-350 DOI: 10.1038/nature07712[↩]

The immune system needs to be rigorously controlled, lest it break its banks and flood the body with destructive responses. Any immune stimulant carries its own brakes; a response to an antigen peaks and then crashes as fast as it accelerated. When the brakes fail, autoimmunity and immune-mediated damage can be more lethal than the pathogen.

This gets complicated with chronic infections. Is it better to shut down the immune response, and let the pathogen romp through your body unchecked, or to let the immune response continue, and risk autoimmune disease? In some cases, shutting down the immune response seems to work pretty well; rodents infected with Hantaviruses (see here, for example) don’t try to fight off these viruses very aggressively, and tolerate the persistent infection pretty well (though not perfectly).

In other cases, though, reducing the immune response may be harmful. Hepatitis C virus infection in humans is linked to high TReg levels and to reduced immune response, and that may be one reason why it persists.

(By the way, it’s worth spelling out what I mean by “reducing the immune response”. In many cases this is mainly a regulatory T cell (TReg) effect, meaning it’s actually an active suppression of the aggressive immune response. Saying that the immune response is “reduced” or “shut down” really isn’t accurate; there’s still a strong and specific immune response, it’s just that the response has been redirected from attacking the pathogen, to controlling the anti-pathogen response. But it’s easier to say that it’s reduced.)

What category is HIV in? Is disease linked to an overactive immune response, or would cranking up immunity suppress the virus and reduce disease?  It’s been unclear, but the consensus is gradually tipping to the idea that the TReg response in HIV infection is more harmful overall (see this paper¹ and the commentary from the Treatment Action Group blog, for example). A recent paper² from the Emory Vaccine Center in Atlanta answers this more directly for SIV in macaques, which may or may not be a valid model for HIV in humans.

“Exhausted Salaryman” – Hiromy

Cytotoxic T lymphocytes (the antiviral killer cells) in chronic viral infections often are “exhausted”: After a certain period of attack, the CTL become dysfunctional, or at least have reduced function. (I believe, but do not know for sure, that this is related to the concept of “polyfunctional” CTL that has recently become popular — control of HIV is correlated with CTL that can produce many different antiviral reagents, while uncontrolled HIV is correlated with CTL that only have one or a few of these reagents. See this post for a little more on that.) This “exhaustion” concept is relatively new, and it’s only in the past couple of years that physical markers of exhaustion have been identified. One such marker is the PD-1 (Programmed-Death-1) molecule,³ and in fact PD-1 is upregulated on CTL during HIV infection.⁴

PD-1 is not a mere passive flag. It’s a receptor that actively drives cells into an inhibited state. If PD-1 is the major reason that CTL are dysfunctional in HIV infection, then perhaps suppressing PD-1 will regenerate the immune response and shut down HIV. Or, of course, it could crank up all the immune responses including those you want shut down, and could lead to a massive and fatal autoimmune attack.

With the SIV/macaque model — amazingly enough; there hasn’t been a lot of good news on the HIV front for a while — blocking PD-1 actually worked just as you’d want it to. After blocking PD-1, anti-SIV CTL frequency roared up,  doubling the pre-treatment levels within a couple of weeks.

After PD-1 blockade, the Gag-CM9 tetramer-specific CD8 T cells expanded rapidly and peaked by 7-21 days. At the peak response, these levels were about 2.5-11-fold higher than their respective levels on day 0.²

And these newly abundant CTL were polyfunctional; they were far more likely to express multiple cytokines than the CTL pre-treatment. Remembering that polyfunctional CTL are correlated with control of HIV, it wasn’t so surprising that after PD-1 treatment SIV levels dropped dramatically after treatment as well. Most impressively, all the treated monkeys survived for at least 150 days, while 4 of the 5 control-treated macaques had died by then (see the survival curve to the left here).

Critically, the PD-1 treatment didn’t cause any side-effects in these monkeys, so at least over this relatively short period autoimmunity wasn’t a problem. Of course, HIV in humans is not exactly like SIV in macaques, and if it turned out to be necessary to have long-term treatment it may be a different story. Even if autoimmunity doesn’t develop, we think that having too many activated CD4 T cells (as opposed to CD8 T cells, CTL) is a bad thing for HIV patients because it makes the CD4 cells more susceptible to infection; if blocking PD-1 increases CD4 activation it might end up being harmful after all.   On the third hand⁵ it’s conceivable that a short-term treatment might reverse the exhaustion and allow the immune system to control HIV, with no further help, for a long period.

Who knows what’s going to happen when it moves into humans;  but so far at least,  it’s one of the most encouraging anti-HIV findings I’ve seen for quite a while.

1. Regulatory T Cell Expansion and Immune Activation during Untreated HIV Type 1 Infection Are Associated with Disease Progression. Weiwei Cao, Beth D. Jamieson, Lance E. Hultin, Patricia M. Hultin, and Roger Detels. AIDS Research and Human Retroviruses. February 2009, 25(2): 183-191. doi:10.1089/aid.2008.0140. [↩]
2. Velu, V., Titanji, K., Zhu, B., Husain, S., Pladevega, A., Lai, L., Vanderford, T., Chennareddi, L., Silvestri, G., Freeman, G., Ahmed, R., & Amara, R. (2008). Enhancing SIV-specific immunity in vivo by PD-1 blockade Nature, 458 (7235), 206-210 DOI: 10.1038/nature07662[↩][↩]
3. Restoring function in exhausted CD8 T cells during chronic viral infection. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ, Ahmed R. Nature. 2006 Feb 9;439(7077):682-7. [↩]
4. Among several other papers:
   PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, Mackey EW, Miller JD, Leslie AJ, DePierres C, Mncube Z, Duraiswamy J, Zhu B, Eichbaum Q, Altfeld M, Wherry EJ, Coovadia HM, Goulder PJ, Klenerman P, Ahmed R, Freeman GJ, Walker BD. Nature. 2006 Sep 21;443(7109):350-4. [↩]
5. Abuse of mutagenic drugs is a constant problem among scientists[↩]

The Nervous System (Fritz Kahn (1888-1968))

Is “immunodominance” just what you get when you measure the wrong place?

Usually, when you look at T cell immune responses to a virus, they’re pretty strongly biased. That is, although the T cells are theoretically, and often observably, able to recognize a wide range of target peptides, the immune response is strongly focused on just a handful of these peptides, while the remaining pool of potential targets is either ignored altogether or given a cursory glance by a handful of T cells. This phenomenon is known as “immunodominance“, and it’s seen with  immune responses to all sorts of pathogens. In some cases — such as for HIV — it’s likely that a strongly immunodominant response is harmful, because it makes it easier for the infecting virus to mutate away from immune control. But in the vast majority of cases the immune response, be it never so immunodominant, does a perfectly good job of controlling the virus; which is why we’re able to easily control most of the viruses that we’re exposed to.

Usually when you measure an immunodominant response, you’ll take lymphocytes from the most abundant, easily-accessed place you can find. That would be blood, in humans; in mice you’d probably take a spleen or lymph nodes.  Some viruses like to hang out in these places, and these include some of the more popular research viruses.

But most of the viruses we’re exposed to don’t infect blood or secondary lymphoid organs; they infect the lungs, or the skin, or neurons, or some other tissue. When we measure the blood response, we believe we’re measuring a good approximation of the real response ongoing in the infected tissue, but that’s mostly been an assumption, not a demonstrated fact.

Recently there’s been some work starting to feel out how similar the tissue response is to the blood/lymphoid organ response. For example, I talked here about work establishing the timing of immune responses in the lungs, vs. the blood. In this case, the overall patterns were similar, though the details were somewhat different.

But that was only really looking at a fairly big picture — overall patterns. What about specifics of target recognition? In particular, is the immunodominance we measure in the blood what actually happens on the battlefield?

I’m only aware of a couple of studies that look at this at all, and those were mainly as asides, noticed in passing. Yewdell’s group has shown in a couple of paper that  infecting mice with poxviruses by different routes leads to differences in immunodominance:¹

The latter point is underscored by our observation that the ID hierarchy varies with the route of infection, the first observation of its kind to our knowledge. It will be of great interest to determine the underlying mechanism. ²

I’ve been told of unpublished data that show different immunodominant responses between lung and spleen, as well; also with a poxvirus.

But in those few examples, the epitopes were all known ones.  Known epitopes moved up or down a notch or two in the immunodominance hierarchy. A recent paper from Bob Hendricks’ group³ shows that T cells in the tissues can recognize things that are apparently not seen at all in the blood or spleen.

Electron tomogram of HSV4

Here they used herpes simplex virus (HSV) in C57BL/6 mice, which have long been believed to almost entirely focus their CD8 T cell response on a single peptide. Hendricks’ group has been looking at the immune response to HSV in the brain, where the virus sets up a latent infection  (I’ve talked about some of his findings here and here).  Contrary to more traditional concepts, it’s now becoming clear (from Hendricks’ work, and that of others) that T cells in the brain are important in controlling latent HSV infection.

In this paper, he found that the immune response in the brain is much more diverse, fairly strongly recognizing at least one  peptide other than the known dominant job.  Because the “normal” (that is, non-neuronal) immune response is so focused, this almost certainly means that the active immune response, down at the pointy end where the T cells are actually working, are responding to altogether different peptides.

It’s generally been assumed, as I say, that the easily-accessed blood or secondary lymphoid tissue is a reasonable approximation of what’s going on in the actual sites of action, in the peripheral tissues — in other words, the idea has been that there’s more or less equal flow of cells between the tissues and the blood and lymph. The recent work on timing and kinetics that I mentioned here sort of supported that assumption, but now we have to wonder whether in fact there’s some kind of filter that keeps some sets of T cells from entering, or staying in, the blood.

We also have to wonder if the whole “immunodominance” paradigm is what we think it is. Could immunodominance represent the filter between blood and tissues, rather than the actual formation of responses? I actually don’t think that would explain immunodominance in general (for one thing, we see strong immunodominance for viruses of lymphocytes, where the blood is the site of infection, so there shouldn’t be a filter) but it’s something to factor in.

1. D. C. Tscharke (2006). Poxvirus CD8+ T-Cell Determinants and Cross-Reactivity in BALB/c Mice Journal of Virology, 80 (13), 6318-6323 DOI: 10.1128/JVI.00427-06
   D. C. Tscharke (2005). Identification of poxvirus CD8+ T cell determinants to enable rational design and characterization of smallpox vaccines Journal of Experimental Medicine, 201 (1), 95-104 DOI: 10.1084/jem.20041912[↩]
2. D. C. Tscharke (2005). Identification of poxvirus CD8+ T cell determinants to enable rational design and characterization of smallpox vaccines Journal of Experimental Medicine, 201 (1), 95-104 DOI: 10.1084/jem.20041912[↩]
3. B. S. Sheridan, T. L. Cherpes, J. Urban, P. Kalinski, R. L. Hendricks (2008). Reevaluating the CD8 T cell response to HSV-1: Involvement of CD8 T cells reactive to subdominant epitopes Journal of Virology DOI: 10.1128/JVI.01699-08[↩]
4. Electron tomogram of a HSV nucelocapsid completing envelopment , from Baines, J. D., C. E. Hsieh, E. Wills, C. Mannella, and M. Marko. 2007. Electron tomography of nascent herpes simplex virus virions. J Virol 81: 2726-2735.[↩]

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 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[↩]

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).

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[↩]

We spend a lot of time trying to understand immune responses against the most virulent pathogens. Perhaps it’s just as useful to look at the response to feeble, marginal pathogens. Serious pathogens are serious because immunity doesn’t control them well, so if we’re trying to understand effective immunity, why not look at minor infections, where the immune system really works?

Most of us have been infected with respiratory syncytial virus (RSV) as children, but most of us never knew it. It’s one of the myriad viruses that are lumped together as “the common cold”. An infant with a runny nos, a bit of a fever, not feeling quite right — maybe wheezing and not eating well — might have RSV; or she might have something else, too. (Even though the vast majority of infected kids have no real problems, because the virus infects essentially everyone, the small minority of problems add up to a large number — some 100,000 children are hospitalized for RSV-related diseases per year in the US alone.)

Immunity to RSV is often considered to be inadequate,¹ but the fact is that most infections are rapidly eliminated without problems. The “inadequacy” label is probably for two reasons. One is that it’s hard to get long-lasting protective immunity; the immune response that cleared the virus doesn’t necessarily protect against a new infection in a year or two. (This is a common factor among many of the common cold complex, of course, though there are probably many different reasons for that.) The other reason is the lingering memory of the disastrous RSV vaccine that I’ve mentioned here previously. ² There seems to be something of a resurgence of interest in the basic pathogenesis of RSV, and a recent paper³ makes some interesting observations.

One of the general problems with understanding immunity to many viruses — especially human viruses — is access. It’s easy to measure immune responses in the blood, because blood is easy to access. It’s not so simple to look at the actual site of infection, whether it’s liver, lungs, gut, or whatever, and so blood is often used as a surrogate. But it’s an open question how closely the immunity in the blood tracks the immunity at the local site. (Again, this is especially true of humans. In mouse studies, you can sacrifice the mouse and remove the lungs. That’s not a real option for human viruses. It’s also an open question as to how well the mouse and human compare.)

In this study, Heidema et al. managed to look at the local lung immune response to RSV, as well as to influenza virus, and compared the local and blood immune parameters. They used tracheostomy patients so that it was relatively easy to access the lungs; easier than running a hose down your nose and washing the bronchi that way, anyway.

Encouragingly, they saw the same general patterns as with mouse experiments. The lung response wasn’t quite the same as the blood. In the lungs, there’s a 3-part response: First, the T cells that are already present in the lungs respond. These are memory cells. We know that memory cells live for a long time, and it was already known that most of the lymphocytes that hang around in the lungs normally are memory type cells:

… long after clearance of a respiratory infection, cells present in tracheal aspirate are of the effector/memory type. These cells reflect the effector/memory cells already present before the next exposure to a respiratory pathogen. ³

Second, both specific and non-specific T cells from the blood enter the lungs. (There’s no way for the circulating T cells to tell which virus is causing the inflammation in the lung, so all of the memory cells drop in to check it out.) The specific ones stick around for a while; the non-specific ones don’t. In mice this seems to be a one-way street, with the non-specific lymphocytes mostly dying off, but from the work here, it’s possible that in humans the non-specific lymphocytes can return to the blood and continue their surveillance.

In the third wave, newly expanded virus-specific T cells enter the lungs. These are the guys who ran into antigen in the draining lymph nodes, got stimulated, divided and became activated, and then went looking for trouble. Because there are a number of events that have to happen before these cells arrive (the antigen has to move from the lung to the draining lymph nodes; the lymphocytes have to respond and dive, and then have to enter the circulation and finally enter the lungs) it takes longer for these cells to appear, but once they’re in they stick around for a long time. In fact, they’re the cells that remain in the lungs to act as the first wave for the next infection.

The experiments are not perfect, given the usual problems of dealing with humans, but there’s a lot of information there, and it should be possible to build on this to figure out more about the local immune response to viruses.

1. ”Unfortunately, RSV infection provides only limited immune protection to reinfection, mostly due to inadequate immunological memory”" — S BUENO, P GONZALEZ, R PACHECO, E LEIVA, K CAUTIVO, H TOBAR, J MORA, C PRADO, J ZUNIGA, J JIMENEZ (2008). Host immunity during RSV pathogenesis International Immunopharmacology, 8 (10), 1320-1329 DOI: 10.1016/j.intimp.2008.03.012[↩]
2. Actually, maybe I’ve never mentioned it, or if I have I can’t turn up the post. I should talk about it, because it’s a fascinating story.[↩]
3. Heidema J, Rossen JW, Lukens MV, Ketel MS, Scheltens E, Kranendonk ME, van Maren WW, van Loon AM, Otten HG, Kimpen JL, van Bleek GM (2008). Dynamics of human respiratory virus-specific CD8+ T cell responses in blood and airways during episodes of common cold. J Immunol., 181 (8), 5551-5559[↩][↩]

“Death and the Doctor” Published by William Humphrey, 1777

Last April I commented on a series of experiments  that used intravital microscopy to visualize cytotoxic T lymphocytes (CTL) attacking a tumor. ¹ Immensely cool though the movie is, I noted that I was surprised by their estimate of the rate of cell killing:

Another surprising finding — which is so different from previous work in different systems that I’m hesitant to believe it — is the timing of cell killing. Previous studies (such as the von Andrian paper² that produced this video) have suggested that CTL kill their targets in something under an hour; maybe 30 minutes or even less. Here. Bousso’s group find that the tumor cells take something like 6 hours to be killed. That’s such a large difference — and has such important implications for effectiveness of CTL killing — that, as I say, I’d like to see it confirmed before I take it to the bank.³

A new paper⁴ has run another estimate of the time it takes for a CTL to kill its target, and like most of the previous work, they conclude that it takes about a half-hour, give or take, to kill a target. They do come up with a fairly wide range of killing times, that depend on the target and the timing of the immune response — at the peak of the immune response when there are many cells the targets are killed faster (between 2 and 14 minutes), while at later stages, when there aren’t so many CTL, targets have half-lives of 48 min and 2.8 hr.

CTL killing a target cell (From a video by von Andrian)

This is not quite looking at the same thing as the video showed, though. In this paper, they were looking at the bulk effects, and that’s what almost all the previous studies have also looked at. The video was looking at a one-on-one interaction. What if targets are killed faster when several CTL gang up on them? Here, having different numbers of CTL caused the half-life of the targets to increase between about 10 and 20-fold. But this is probably simply because, with fewer CTL present, it took longer for them to find the target: Once a CTL found the target, the rate of killing was if anything faster than effectors at killing (”we find that LCMV-specific memory CD8 T cells kill more target cells per day than effectors”). ⁵

This is actually a disagreement with a previous paper ⁶ that also looked at killing rates, and offered evidence that different types of CTL can have different killing rates:

We reanalyse data previously used to estimate killing rates of CTL specific for two epitopes of lymphocytic choriomeningitis virus (LCMV) in mice and show that, contrary to previous estimates the “killing rate” of effector CTL is approximately twice that of memory CTL. ⁶

However, whichever of those studies is correct , both suggest that different types of CTL can have different killing efficiencies. This goes back to a point I’ve made several times, as have others (see e.g. Michael Palm’s TAG post here and references therein, including the comments by me and by Otto Yang) — CTL aren’t a uniform batch, and different kinds of CTL may have different types as well as rates of activities.

Returning to the intravital microscopy killing rate of 6 hours:⁷ I wonder if that reflects the nature of the CTL there, perhaps influenced by the tumor environment. Tumors are notoriously resistant to killing (probably because those tumors that are not resistant to killing were, um, killed, before they ever become clinically detectable) and it seems quite likely that an immunosuppressive tumor environment may change CTL types, or activities. I wonder if that would offer some way of intervention. Speeding up the rate of CTL killing from 6 hours to 30 minutes seems like it would be a huge influence of clearance of tumors. On the other hand, of course, it may be that the targets themselves are much more resistant to killing (again because tumor cells have been through selection to be resistant to the immune system) and cranking up CTL won’t make much difference.

1. Breart, B., Lemaître, F., Celli, S., Bousso, P. (2008). Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice. Journal of Clinical Investigation, 118(4), 1390-1397. DOI: 10.1172/JCI34388 [↩]
2. Mempel, T. R., Pittet, M. J., Khazaie, K., Weninger, W., Weissleder, R., von Boehmer, H., and von Andrian, U. H. (2006). Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation. Immunity 25, 129-141.[↩]
3. From this post[↩]
4. V. V. Ganusov, R. J. De Boer (2008). Estimating In Vivo Death Rates of Targets due to CD8 T-Cell-Mediated Killing Journal of Virology, 82 (23), 11749-11757 DOI: 10.1128/JVI.01128-08[↩]
5. There are also other videos of one-to-one killing, at least in vitro, that are more consistent with the 30-minute ballpark; see the image to the right for one example.[↩]
6. Yates A, Graw F, Barber DL, Ahmed R, Regoes RR, et al. (2007) Revisiting Estimates of CTL Killing Rates In Vivo. PLoS ONE 2(12): e1301. doi:10.1371/journal.pone.0001301[↩][↩]
7. Which I have become more relaxed about since my earlier skeptical comment[↩]

HIV budding from a macrophage

The STEP anti-HIV vaccine trial  received a lot of press coverage last year, when the vaccine was pulled for fear that it actually worsened HIV disease. A number of mechanisms were proposed for the exacerbation.  One of those has now received some support.¹

The STEP study used adenovirus vectors, expressing HIV proteins, to induce immunity to HIV. Adenoviruses are ubiquitous viruses in most human populations, usually causing fairly mild upper respiratory tract infections (i.e. cold-like symptoms), and most people have been repeatedly exposed to adenoviruses. As part of the adenovirus/HIV vaccine, people developed immunity to the HIV proteins, and also increased their immunity to the adenovirus component. Unfortunately, the preliminary analysis suggested that those vaccinees with high anti-adenovirus immune responses, were actually more susceptible to HIV, not more resistant. Obviously, that was a bad thing.

One suggestion at the time was that having immunity against adenoviruses might lead to increased activation of the immune system. There was already evidence at the time that activated T cells are more susceptible to HIV infection in several ways, and that evidence has been boosted by several studies since. For example, just the other day there was a paper showing that

… circulating microbial products can increase viral replication by inducing immune activation and increasing the number of viral target cells, thus demonstrating that immune activation and T cell proliferation are key factors in AIDS pathogenesis.²

In fact, in monkey species (e.g. sooty mangabeys) that don’t develop disease after SIV infection, you don’t see a lot of immune activation; whereas those species that do develop disease, show significant immune activation:

SIV-infected SMs³ do not manifest the chronic generalized immune activation that characterizes pathogenic SIV and HIV infections, a process that is thought to play a central role in driving CD4+ T cell depletion through bystander activation and loss of uninfected T cells. ⁴

HIV infecting a macrophage5

So there was theoretical support for the concept that immunity to adenoviruses could lead to immune activation, which in turn could lead to increased HIV replication, causing increased susceptibility to HIV. The paper I mentioned that provides more direct support ¹   also spells out a mechanism in a little more detail, looking at antibodies against adenovirus and their effect on activation; as well as noting that at least one other potential problem might be that the anti-adenovirus response could indirectly cause a reduced anti-HIV response (by killing dendritic cells).

This study (and others) actually point to a couple of useful directions. For one thing, although in this case it seems that immune activation was bad, in most cases it’s just the opposite.⁶  The anti-HIV vaccine is actually a very special case where we might not want an activated immune response, and even there it’s not strictly activation we want to avoid, just off-target activation. (A strong, activated immune response against HIV is probably a good thing,⁴ because it can shut down the virus.) This adenovirus trick may be a fairly straightforward way of getting immune activation, if it can be harnessed.

Another point is that in this special case, where immune activation may be harmful, maybe blocking activation would be beneficial. It’s a little counterintuitive to try to suppress the immune response when you’re infected with a virus, but it’s probably worth looking at:

These data suggest that therapeutic strategies to reduce immune activation should be explored, in addition to the classic antiretroviral therapies, in preventing progression to AIDS in chronically HIV-infected individuals.²

Added note: The Michael Palm Treatment Action Group blog has commentary on the STEP vaccine trial conclusions published in The Lancet, as well as a previous series of commentaries on the vaccine trial. Highly recommended.

1. M. Perreau, G. Pantaleo, E. J. Kremer (2008). Activation of a dendritic cell-T cell axis by Ad5 immune complexes creates an improved environment for replication of HIV in T cells Journal of Experimental Medicine DOI: 10.1084/jem.20081786[↩][↩]
2. The Journal of Immunology, 2008, 181: 6687-6691.[↩][↩]
3. SMs: Sooty Mangabeys[↩]
4. J Immunol. 2008 May 15;180(10):6798-807[↩][↩]
5. Gross, L., 2006. Reconfirming the Traditional Model of HIV Particle Assembly. PLoS Biology, 4(12), p.e445 EP [↩]
6. Bad.[↩]