Rube Goldberg machineAntigen processing is not only interesting and important in itself,1 but it’s been used extensively to tease apart fundamental cell biology — things like protein folding, intracellular proteolysis, protein trafficking, and ER-associated degradation have been identified or studied via antigen processing. There are a bunch of reasons why MHC has been such a Swiss army knife of cell biology. One of the reasons is that MHC can amplify a tiny, tiny signal into a blatant, unmistakable readout.

That’s because cytotoxic T lymphocytes recognize MHC/peptide combinations, recognize it incredibly well, and respond with easily-observed events. CTL can recognize as few as 10 (maybe fewer) specific peptides per cell, even though for every one of those peptide/MHC complexes there are ten thousand other complexes with other peptides, smothering it. And CTL respond by destroying the cell, which gives you a simple, black-and-white, binary outcome.

It’s obviously useful to have a highly sensitive2 readout. But it’s a curse as well as a blessing. In particular, because the outcome is binary (alive or dead) it’s really hard to get quantitative information out of the system. Once you’re over the very low threshold, everything is positive.

What this means is that detecting something with a CTL readout doesn’t tell you if that something is common, unusual, rare, or sui generis. CTL readouts over the years have demonstrated the existence of events that (I believe) are really very unusual — they aren’t representative of “normal” cell biologic processes, but rather represent the far end of the curve, things that, yeah, can happen, but have to be pushed. For example, there’s proteasome splicing : biochemically a really cool phenomenon, that got picked up by CTL readouts — but it’s really not likely that it happens very often, or is a real player in the normal function of the cell.3

On the other hand, of course, some things that have turned out to be common and important processes were identified up in the same ultrasensitive way. For example, exactly this sort of thing turned out to be a very early demonstration of ER-associated degradation,4 which is now known to be a major and critical pathway.

HIV-1 frameshift inducing element
HIV-1 frameshift inducing element

So — following on from my post earlier this week — the immediate question that comes to mind when Nilabh Shastri’s group publishes about frame-shifted epitopes5 is whether this is a major, common phenomenon, or it is the end-product of a Rube Goldbergesqe sequence of events that isn’t going to happen very often?

Shastri has long been a fan of the idea that frame-shifting — reading proteins from abnormal start sites, or by hiccups during translation — could be a common source of antigenic peptides (epitopes). In his latest paper, he demonstrates a frame-shift epitope from HIV; he and some other groups have demonstrated frame-shift epitopes before, but those were mostly fairly minor, and were easy to ignore. This example seems to be a relatively potent epitope, and is harder to ignore. Are frame-shifts common in the cell? Are they common sources of CTL epitopes?

ResearchBlogging.orgInterestingly, they identified the epitope by bioinformatic analysis of a known frame-shift product. (In other cases, the identification went the other way around, from identifying the epitope sequence to the frame-shifted precursor.) This raises one point: If frame-shifted proteins really are common sources of CTL epitopes, then for one thing the task of the bioinformatician is six times harder, because they will have to survey all six reading frames, not just the known proteins, of a viral genome, to look for epitopes. But (for all the criticism I’ve leveled at epitope prediction software) that doesn’t seem to be a major factor; predictions do find epitopes (however inefficiently) and they find them in true proteins.

In the small handful of cases where a full CTL response to a virus has been analyzed fairly completely — that is, where almost all the epitopes recognized the CTL have been identified — they almost all have been identifiably from authentic viral proteins.6 That said, there are some that haven’t been identified yet; for example, in mouse cytomegalovirus a number of epitopes remain unmapped,7 and might be from frame-shifted precursors.

Proponents of the unconventional precursors argue that many MHC-associated peptides (identified by mass spectrometry, for example) don’t have an authentic protein precursor in the various databases. I think that’s true, but far more are identifiable (I don’t know the ratio of identifiable to unidentifiable, though), and most of the anonymous ones probably represent, say, un-sequenced alleles or something like that.

Overall, I think the bulk of the findings from epitope identification really argue that things like frame-shifted epitopes, or proteasome-spliced epitopes, or non-ATG-initiated epitopes — things that we think should be rare, based on what we know about cell biology — really are rare. The fact that they do appear and can be captured by this exquisitely sensitive8 system, probably goes to show that there is more slop in the system than is often believed — more aberrant, defective products sneak through into RNA and protein than is really appreciated, and in all likelihood error correction is just as important as error prevention in normal cell function.

  1. And people who research antigen processing are invariably suave, attractive, and charming. Well-known fact![]
  2. I’m desperately trying to avoid saying “exquisitely sensitive” here, because every paper and review on the subject calls it “exquisitely sensitive”[]
  3. I reserve the right to deny I ever said this, if proteasome splicing ever turns out to be important.[]
  4. Skipper, J. C., Hendrickson, R. C., Gulden, P. H., Brichard, V., Van Pel, A., Chen, Y., Shabanowitz, J., Wolfel, T., Slingluff, C. L., Jr., Boon, T., Hunt, D. F., and Engelhard, V. H. (1996). An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. J. Exp. Med. 183, 527-534.[]
  5. Maness, N. J., Valentine, L. E., May, G. E., Reed, J., Piaskowski, S. M., Soma, T., Furlott, J., Rakasz, E. G., Friedrich, T. C., Price, D. A., Gostick, E., Hughes, A. L., Sidney, J., Sette, A., Wilson, N. A., and Watkins, D. I. (2007). AIDS virus specific CD8+ T lymphocytes against an immunodominant cryptic epitope select for viral escape. J Exp Med 204:2505-2512 []
  6. Kotturi, M. F., Peters, B., Buendia-Laysa, F. J., Sidney, J., Oseroff, C., Botten, J., Grey, H., Buchmeier, M. J., and Sette, A. (2007). The CD8+ T-cell response to lymphocytic choriomeningitis virus involves the L antigen: uncovering new tricks for an old virus. J Virol 81, 4928-4940. []
  7. Munks, M. W., Gold, M. C., Zajac, A. L., Doom, C. M., Morello, C. S., Spector, D. H., and Hill, A. B. (2006). Genome-wide analysis reveals a highly diverse CD8 T cell response to murine cytomegalovirus. J Immunol 176, 3760-3766.[]
  8. Couldn’t keep it up[]