Tuesday, 19 February 2013

T-Cell Receptors, Part Two: A History of TCR Discovery

Back to Part 1: The immtroduction
Nowadays we know quite a lot about TCRs. We know the general mechanics of how they're made, what they respond to, and how they respond to those things (note the 'general' - all three of these are vastly complicated processes, which are still topics of intense research). However, as is the way with all knowledge, this was not always the case.

The first appearance TCRs make into the literature (that I'm aware of) occurred back in the tail end of 1982. Researchers from Texas (among them, apparently, a member of the botany department, which is nice) were looking to generate antibodies against specific mouse T-cell lymphoma clones.

They constructed a library of B-cell hybridomas and immunised them with a model lymphoma line from a different mouse strain, before screening for antibodies that would recognise just the lymphoma cells. They found one; their antibody only reacted with the intended lymphoma; other lymphomas, T-cells, or spleen cells weren't targetted.

Pulling down the target by radioimmunoprecipitation (which is such an old and risky immunoprecipitation technique, it's not even listed on the wikipedia page) revealed a disulphide-linked glycoprotein heterodimer.

More importantly, examination of purified T-cells (but not B-cells) showed that other T-cells had similar structures on their cell-surfaces, but these weren't reactive against their new antibody. Sounds like we have a clonal marker of T-cells here!

The next year saw a flurry of papers: the finding was repeated in humans, and evidence piled up to show that this was the variable, clonotypic, T-cell specific receptor in both humans and mice. It wasn't called it yet, but the αβ TCR had been found.

Next came the laying of the groundwork for my project (which I assume is why they did it); the search for the genes that encode these receptors. The majority of the TCR genes were cloned through a nucleic acid subtraction strategy, which was based upon a few assumptions about the nature of the TCR genes, such as:

  • they will be expressed by T-cells, but not B-cells
  • they will likely be rearranged, as B-cell antigen receptors were already known to be
  • in addition to their variable region (for antigen binding), they should have a constant region (to mediate downstream effects of antigen binding).

With these concepts in mind, the substraction strategy proved to be a pretty cunning choice. Total RNA was extracted from antigen specific, T-cell hybridomas, and reverse transcribed into first strand cDNA, radiolabelled with 32P. Then second strand cDNA (i.e. the same orientation as the sequence encoded in the RNA) is prepared from another closely related, but non-identical source (some papers used B-cell hybridomas, while others opted for different T-cell hybridoma clones), which is then incubated with the labelled T-cell cDNA.

Any sequence that is shared by both cell types will hybridise together (as the prepared cDNA for each was of the opposing strand). Any non-shared sequence remains single stranded, and can be selected out by hydroxyapatite fractionation; the shared transcripts get subtracted from the T-cell specific ones.

Using this technique, the β chain genewas cloned first. The next gene cloned was assumed to be the pair of the β, which would make it what we now call the α chain. However, partial protein sequences from the binding partner of β didn't match the translated sequence of this second gene.

What'd they'd actually cloned was the γ chain; it was the third gene cloned that turned out to be the partner of the original, and thus the α chain.

(For those that are interested, there's a great contemporaneous review written by Frank Fitch that sums up pretty much all of what was known about TCRs at the time.)

This left us with a little mystery; if the first TCR gene (β) paired with the third one found (α), what's the second one (γ) doing?

It was a couple of years before the answer came to light, with the discovery of γδ T-cells; non-αβ T-cells which expressed CD3 (or T3, in old money) associated with the product of the TCRγ gene along with the previously unknown binding partner for γ: δ had been δiscovered.

It took a different trick to discover the gene for TCRδ, along with a bit of serenpidity. These researchers were using pulsed-field gel electrophoresis (a technique for the separation of very large DNA fragments) to investigate recombination in the TCRα locus.

In running these huge gels, they noticed that a region ~90kb upstream of the TRAC gene was undergoing rearrangement. Upon closer inspection there were homologous VDJ gene segments, getting recombined together and spliced onto a novel constant region.

It's small wonder the δ locus was so hard to find; it was hiding inside the α locus, making use of members of the same V region array. Plus, it gets completely deleted by any successful α recombination - which incidentally makes for a nice failsafe to ensure an αβ-T-cell can't express any γδ-TCR by mistake.

There we have it; a brief(ish) account of the discovery of all four chains of the TCR (discounting the funny ones that some other animals have). 

So, now we know how and where they're supposed to be expressed, we can start to appreciate the intrigue about when they pop up elsewhere...

On to Part 3, where the TCRs start to roam

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