Cytotoxic T lymphocytes (CTL) recognize peptides that are about 9 amino acids long. There are lots of constraints on which peptides can possibly be presented; the most important factor is whether the peptide can bind to one the MHC class I alleles that the host expresses. Still, a generic virus will have hundreds or more likely thousands of peptides that are reasonable CTL targets. Of those peptides, how many are actually recognized by CTL? Of those that are recognized by CTL, how many are recognized effectively (enough to trigger a detectable response)? Does it make any difference which, and how many, are recognized? And — most interestingly — why are so few peptides recognized?
There are technical problems with this question. One huge problem is just how to identify the peptides that are recognized. Typically, you’d have to synthesize peptides from the viral genome, mix them with CTL from an immune host, and figure out which of the peptides activate the CTL. However, if you try to synthesize all the possible peptides from a viral genome, you’ll have many thousands of peptides: Expensive, to say nothing of the work involved in screening.
People have tried to get around this in two ways. One is to use longer peptides. Traditionally, screening has used 15mers rather than 9mers. Using overlapping 15mers instead of every possible 9mer can cut your screening down into a relatively manageable range — a couple thousand or fewer. Still a big job, but practical. One problem with this, of course, is that 15mers shouldn’t work at all for MHC class I! MHC class I alleles (in contrast to MHC class II) rarely bind peptides anywhere near that long; rarely much more than 11 or so amino acids long. So what you’re counting on, with your 15mers, is that either they’re contaminated with incomplete synthesis products (a common situation), or that they’re partially degraded in the medium when you add them to your cells. In either case, you really don’t have a good idea what your actual coverage of the viral proteome is.
Another approach is to try to cut down your required peptides, by trying to predict which ones could possibly bind to your MHC class I and only (or mainly) synthesizing those. The problem here is that for all the progress in understanding MHC class I binding motifs, there are lots of high-affinity peptides for various MHC class I alleles that don’t even come close to matching the putative binding motif. Your coverage is only as good as your predictions, and your predictions will miss some genuine epitopes.
(Another possible problem with both of these approaches is that they’ll miss peptides that are not part of the viral proteome. That includes things like spliced peptides (see my previous post on that), out-of-frame peptides,1 and post-translationally modified peptides that don’t match the encoded sequence — the most famous example probably being glycosylation sites where the carbohydrate is stripped off the Asn in the cytosol to leave a non-templated Asp.2 )
This brings me to Kotturi et al, a paper I’ve mentioned here before:
The CD8 T-Cell Response to Lymphocytic Choriomeningitis Virus Involves the L Antigen: Uncovering New Tricks for an Old Virus
Maya F. Kotturi, Bjoern Peters, Fernando Buendia-Laysa, Jr., John Sidney, Carla Oseroff, Jason Botten, Howard Grey, Michael J. Buchmeier, and Alessandro Sette
Journal of VIrology, May 2007, p. 4928â€“4940 (doi:10.1128/JVI.02632-06)
Lymphocytic choriomeningitis virus (invariably abbreviated to LCMV for obvious reasons) is one of the classic models of viral immunity. One of its many nice qualities3 is that it induces a tremendous (i.e. easily measured) immune response. At the peak of the immune response, 6 to 8 days after infection, some 80 to 95% of a mouse’s CD8 +ve T cells4 may be reactive with LCMV. That makes it relatively easy to detect individual components of the response. In other words, you can readily define individual peptide epitopes within the CTL response to LCMV. Another nice thing about the virus is that it’ usually cleared, if you infect an adult mouse, so you can then move on to analyze memory responses, but I won’t get into that today. (The image on the left is of an arenavirus [LCMV is in the arenavirus family] from Michael Buchmeier’s lab at Scripps.)
Because LCMV has been studied for a while, and because the CTL response is so large, there have been a bunch of viral epitopes defined; in the commonly-used C57BL/6 mouse, 7 peptides were known to induce CTL reactivity since 1998.5 Seven epitopes is actually a fair number — most viruses don’t have that many defined epitopes for just two MHC class I alleles — but three more epitopes were added earlier this year6 bringing the total to 10 defined epitopes that bind to the B6 mouse MHC class I alleles. (The image on the right shows two of the best-recognized peptides from LCMV glycoprotein, in the shape they assume when bound to particular MHC class I alleles. Taken from: A structural basis for LCMV immune evasion: subversion of H-2D(b) and H-2K(b) presentation of gp33 revealed by comparative crystal structure analyses. Achour A, MichaÃ«lsson J, Harris RA, Odeberg J, Grufman P, Sandberg JK, Levitsky V, KÃ¤rre K, Sandalova T, Schneider G. Immunity. 2002 Dec;17(6):757-68.)
However, these 10 epitopes only account for around 80% of the CTL response to LCMV — that is, if you take all the CTL that light up in response to an authentic LCMV-infected cell, about a fifth of those will not light up in response to any of the known epitopes. What are those remaining guys reacting to? Kotturi et al went looking for the missing triggers.
They used both of the approaches I’ve mentioned here. They not only screened with overlapping 15mers covering much of the LCMV proteome, they used MHC prediction programs to identify particular candidates for CTL epitopes and screened those particularly. All in all, they looked at 1064 peptides: “A total of 400 Kb and Db algorithm-selected peptides, along with a set of 664 15-mer peptides, overlapping by 10 amino acids, spanning the entire LCMV proteome, were synthesized.”
Now, remembering that this is an intensively-studied virus, one that’s been a workhorse of immunology for decades, how many new epitopes do you think they turned up? Ten are already known. Kotturi et al turned up another 19 — they nearly tripled the number of MHC class I epitopes for LCMV. That’s the first remarkable thing; it suggests that probably most claims for the number of viral peptides that are recognized are drastic underestimates. (It also suggests that cross-reactive T cells are not common, but that’s another story.)
The next interesting point about their paper is where they got their hits — from their predicted epitopes, or from their 15mers? Well, the predictions did pretty well:
The 15-mer approach including truncated peptide sets required synthesis and testing of 1,2147 peptides and identified approximately 65.2% of the overall response. By contrast, the predictive approach required synthesis and testing of 400 peptides (or 160 if only the top 1.2%8 from each allele would have been synthesized) and identified approximately 88.9% of the total response.
But the predictions did miss several true epitopes; some of the genuine MHC class I epitopes just don’t look like things that are supposed to bind to H-2Kb. If you want to pick up on things that are not, as yet, predictable, you still need a brute-force approach.
So of the hundreds or thousands of potential LCMV epitopes, there are 29 that actually get recognized.9 That’s a fair number of epitopes. But here’s the next part (in fact, this is the whole point of this post). Look at the distribution of CTL responses to each peptide. Here’s what it looks like as a fraction of the total CTL response to LCMV:
The top 2 peptides of the 29 cover 25% of the response; the top 4, 50%. You need to put the bottom 18 peptides together to catch up to the first two and make up 25% of the response!10 This, ladies and gentlemen, is what we call immunodominance. The top handful of peptides are immunodominant — in a C57BL/6 mouse, those peptides will invariably be the targets of the vast majority of the CTL response.11 The other peptides will cause a response that, while detectable, is much lower than that to the dominant peptides.
Well, we don’t know, but at least we think we know some of the possible explanations. More in a later post.
- Nilabh Shastri has probably been the strongest supporter of this concept. See, for example, Constitutive display of cryptic translation products by MHC class I molecules. Schwab SR, Li KC, Kang C, Shastri N. Science. 2003 Sep 5;301(5638):1367-71. I’m not yet convinced that this is as common as he argues, but it clearly can happen.[↩]
- There are a number of examples of this now. The first demonstration that it can happen was: An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. Skipper JC, Hendrickson RC, Gulden PH, Brichard V, Van Pel A, Chen Y, Shabanowitz J, Wolfel T, Slingluff CL, Boon T, Hunt DF, Engelhard VH. J Exp Med. 1996 Feb 1;183(2):527-34.[↩]
- for an immunologist, anyway[↩]
- Quantitating the magnitude of the lymphocytic choriomeningitis virus-specific CD8 T-cell response: it is even bigger than we thought. J Virol. 2007 Feb;81(4):2002-11. Masopust D, Murali-Krishna K, Ahmed R[↩]
- van der Most, R. G., K. Murali-Krishna, J. L. Whitton, C. Oseroff, J. Alexander, S. Southwood, J. Sidney, R. W. Chesnut, A. Sette, and R. Ahmed. 1998. Identification of Db- and Kb-restricted subdominant cytotoxic T-cell responses in lymphocytic choriomeningitis virus-infected mice. Virology 240: 158â€“167.[↩]
- Masopust, D., K. Murali-Krishna, and R. Ahmed. 2007. Quantitating the magnitude of the lymphocytic choriomeningitis virus-specific CD8 T-cell response: it is even bigger than we thought. J. Virol. 81:2002â€“2011. Yes, same reference as before, but I can’t bear to struggle with these footnotes any more.[↩]
- The 664 was their starting pool of 15mers; to actually find the epitopes, they had to synthesize sub-peptides from within the positive 15mers.[↩]
- They broke down the success rate by the rank of the prediction and found that in fact they could have covered most of their hits by using fewer peptides from the most confident predictions[↩]
- There may even be a handful of others; Kotturi et al. don’t account for a few percent of CTL responses even with all the known epitopes. But that may be a sensitivity issue, so let’s assume that the 29 cover everything[↩]
- So, even if the missing few percent of responses are real, one would expect that it would be divided up among many — dozens? Hundreds? — of individual peptides, perhaps all below the limits of sensitivity for present assays.[↩]
- As a side note, even though the predictions did reasonably well — surprisingly well, to me — within their predictions the rank wasn’t a good correlation of immunodominance. For example, the most dominant peptides (50% of the total response) ranked 2, 25, 14, and 28 as predicted epitopes, whereas the three peptides with the highest prediction rank only covered 3.5% of the total response all together[↩]