T-Cell Receptors, Part Five: A Receptor For All Cells?

Back to Part 4: More Odd TCR Transcripts

This last installment in this week of blogging covers the more recent, and potentially more controversial stories I've found of wandering TCRs.

The claims made in the following papers are big, and could have far reaching connotations if they bear out. All of these papers report whole, functional TCRs, being expressed and used by white blood cells. Just the wrong ones.

It all starts in 2006, with a sentence. A tantalising, if near-painfully vague sentence:

"A series of control experiments prompted us to test the hypothesis that human neutrophils express components of the TCR machinery."

A series of control experiments ey, that old chestnut.Wait, what, neutrophils?

These cells are one of the major components of the innate immune system, and account for the majority of circulating white blood cells at any one given time, despite their rapid turnover.

Their job is to turn up early to places where the body is in trouble (such as inflamed or infected areas) and do some damage control, which largely consists of eating any pathogens they can find, and making it harder for any they can't find to survive or spread.

Previously they'd been thought to act completely via innate immune receptors. The presence of one of the hallmark receptors of adaptive immunity on them is a little surprising to say the least.

Using antibodies directed against the alpha and beta constant regions, they report that around five to eight percent of freshly isolated neutrophils express αβ-TCR, seemingly in a manner comparable to typical TCR expression.

They seem to express a varied repertoire, when looking for different Vα and Vβ transcription by RT-PCR, that are revealed to be rearranged as per normal. There's CD3 components, CD28, all seemingly upregulated by TCR agonists, as well as a number of proteins required for TCR signalling.

So what are these TCRs supposed to be doing in these neutrophils? Upon TCR stimulation (with anti-CD3 and anti-CD28 antibodies, which is thought to represent a fairly physiological level of stimulation), and watched what the neutrophils did next.

What they did next was live long and prosper; it seemed stimulating the TCRs inhibited neutrophil apoptosis and increased secretion of IL-8, the chemokine responsible for recruiting more neutrophils to the danger zone.

So, the theory goes that some neutrophils express TCR, which presumably helps them recognise either specific pathogens, or a broader swathe of pathogens, which can then find bugs quicker, helping to recruit more neutrophils to the threat.

The same group (Wolfgang's Kaminski's group from Heidelberg) has had a couple of follow up papers on this, during which time it gets re-dubbed the TCRL_n, as it's TCR-'like', and in neutrophils, which somewhat solves the quandry of the 'TC' in TCR.

One of these papers is really just a long observation that the repertoire of different TCRs expressed in neutrophils starts off broad, and shrinks with increasing age.

The next is just a case study of a patient with a nasty autoimmune condition (where their red blood cells get targetted and destroyed by their own antibodies), whose number of TCR positive neutrophils had jumped from 5% to 80%.

There's also one final neutrophil paper from a dental group, who noted that oral neutrophils also seem to have a higher expression of TCR than do their circulating cousins, which ties in with their previous work showing that oral neutrophils have a different phenotype.

It takes three years after the start of the neutrophil story before the next cell joins the party. Eosinophils, a kind of granulocyte (mostly responsible for killing parasitic worms) enter the fray, wielding not αβ, but γδ-TCRs.

It's another frustrating start; this time they were on the hunt for γδ TCRs in eosinophils because of the "surprising similarities" between them. Either this is fantastically lucky fishing, or there's a few experiments they're not telling us about.

Either way, they find the γδ-TCR on the eosinophils by flow, along with CD3. Interestingly, they don't find much γδ in lymphocytes (1.4%, which is a few percent shy of usual), nor do they find αβ expressed on neutrophils, putting them at odds with the previously discussed papers.

It seems that while possessing all the bits and bobs needed for TCR recognition, these eosinophils don't produce nearly as much TCR message as T-cells, nor as diverse a range of TCRs. However, activation with TCR agonists caused eosinophils to do exactly what they're supposed to do when activated normally, with degranulation and the release of cytotoxic proteins and reactive oxygen species (ROS).

There's even a couple of figures showing exploring possible functions for the γδ receptor. The presence of γδ-blocking antibodies inhibits the ability of eosinophils to produce ROS in response to mycobacterium, or to induce apoptosis in a colorectal cancer cell line.

The final additions to the TCR club is reported by the same Heidelberg group, giving the impression they probably went on a TCR-testing rampage.

Macrophages are key cells responsible for maintaining immunity in the tissues, by phagocytosing and killing pathogens and presenting their antigens to T-cells. They differentiate from monocytes, which circulate in the body looking for signs for infection or inflammation.

These are classical examples of innate immune cells that bridge the gap to adaptive immunity, through their antigen presentation. These recent papers suggest that they might go one further, with some subpopulations expressing either the αβ or γδ TCRs.

The story unfolds much like the others. There's a small percentage of monocytes and macrophages expressing a limited repertoire of 'TCRL_m' (5% for αβ, 3% for γδ), along with other TCR signalling components.

The obligatory search for function touches on some big topics.

For the TCRL_mαβ, they investigate the intereaction with mycobacteria, which it seems upregulate the expression of the TCR. They go further, and stain lung sections from tuberculous patients, showing that the contacting edge of cells around the granulomas are enriched with TCR expressing macrophages.

TCRL_mγδ on the other hand was investigated during (murine) bacterial meningitis, during which the presence of γδ-macrophages was enriched in the CSF. They also found γδ-MΦs in atherosclerotic plaques, indicating a pretty broad range of possible interactions.

There's a lot to muddy the water in these papers. There's a couple of not-completely-convincing figures, and the obvious matter of the contradiction between the papers. They do all however go a long way to refute the possibility they're just seeing T-cell contamination (either by immunohistochemistry or FISH).

These papers seem to ask more questions than they answer. How does this happen. Do they undergo any selection, and if not, how do they avoid auto-reactivity? If so many other cells can make use of the TCR, why do we even need T-cells in the first place?

If true, these are hugely interesting findings. Here we have terminally differentiated myeloid cells, seemingly expressing one of the classical lymphoid, adaptive immune receptors. Having presented some of these at journal clubs I've seen first hand a bit of resistance to accepting these possibilities outright.

Personally, my view is that biology is a massively confusing thing, and the closer we look at it the more wierd, unexpected stuff we're going to find. Once biology has evolved a system such as the TCR, there's no reason why other cells within the same organism shouldn't make use of it. As technology increases the throughput and sensitivity at  which we can operate, more and more of our accepted models are going to gather inconvenient aberrations like these.

There we have it; the case of the wandering TCR. Whether or not you believe these stories (or more importantly, whether or not you think they have any biological signnificance), I hope you found them as interesting as I did.

While this week of blogging ballooned into a much bigger project than intended, I've enjoyed doing it. Over the coming months I hope to do some other key aspects of TCR biology, just maybe not all at once next time.

T-Cell Receptors, Part Four: More Odd TCR Transcripts

Back to Part 3: TCRs Where They Shouldn't be 

After the discovery of TARP, things were a quiet for a little while; it looked like TCRs seemed happy to remain in the T-cells. However, there were a couple of blips on the radar, around the same time, that never really developed into fully-fledged stories.

The first of these two papers was in 2002, working off the back of an interesting observation from a previous study; when analysing the expression of TCR expression in T-cells that adhere to stromal bone marrow cells, they found that the control group of stromal cells alone seemed to express TCR as well as the T-cells (NB, in mouse) .

Closer investigation revealed that mesenchymal cells expressed αβ TCR (both primary cells and cell lines) mRNA, as well as CD3, revealed by RT-PCR. However, neither of the RAG proteins are expressed, and what little TCR there is expressed (by T-cell standards) isn't re-arranged, which you'd expect without the necessary recombinases present.

Once again we see that the transcripts are made of (different) J regions correctly spliced onto constant regions, akin to what we saw with TARP. As the whole constant region was present, they were afforded the luxury of easily looking for protein on cells, as constant region antibodies exist; lo and behold MEFs (as a model mesenchyme system) stain positive for TRBC, while MEFs from TCRβ-/- mice don't (although no T-cell staining as a positive control? Seriously?).

Curiously, mesenchyme cell lines that expressed more of the TCR (at RNA level) seemed to have different growth properties to those that didn't, both proliferating more and causing a greater incidence of cancer when injected intradermally into nude mice.

The next case we see of wandering TCRs takes us to the relatively young field of neuroimmunology.

While we all know and appreciate the role MHC proteins and CD3 play in immunology, it turns out they're required for a number of neurological roles, such as correct synaptic formation and hypothalamus development.

This, and similar observations, prompted Harvard researchers to wonder if perhaps there might be some TCR lurking in the CNS, to act as the ligand for these seemingly important neuronal MHC proteins.

A series of in situ hybridisation experiments shows that there was indeed TCRβ expressed in brain segments, and that it is indeed localised to the neurons. Examination of the message revealed a curious thing about the transcripts (and I think you've guessed it); they're non-recombined, consisting of a (specific) J region spliced to the constant region.

They find no TCRα mRNA, and detect no constant region protein by immunostaining. In addition, TCRβ knock out mice fail to show a similar neurological phenotype to MHC or CD3 knock out mice, which seems to put paid to the idea that this neuronal TCRβ expression provides the ligand for MHC in the CNS.

The authors offer a few suggestions for why the CNS might be expressing this TCR; maybe the RNA serves a purpose, maybe the protein or activity is just very low and undetectable, or maybe the expression is just part of some vestigal transcriptal program, playing no role but doing no real harm. Not the most earth-breaking of papers, but another curious case of TCR message getting around and seeing the sights.

(As a brief aside, this reminds me of an interesting neuroimmunology paper I saw a little while ago, where a genome wide investigation into narcolepsy revealed the major associative polymorphisms mapped to the TCRα locus. This was only an association study, but it does speak to possible non-immune roles for the TCR.)

Both of these stories are a little reminiscent of the B-cell TCR expression we saw in part 3, expression of funny TCR transcripts (spliced but lacking Vs), in cells that we wouldn't expect them in. Interestingly, there's even a link to increased cell proliferation, which echoes the TARP story somewhat.

However, there's very little (at present) to convince me these transcripts are doing much, if anything. Personally, from my own experience sequencing TCR repertoires, I know in T-cells we get a bunch of odd transcripts popping up, the type of which have been known about for a long time. It's always hard to say whether these things biologically important or just interesting looking enough to give postgrads false hope.

Chances are good they just represent the noise in the machine, little ripples of expression as a stone is dropped nearby in the transcriptional pond, unintended byproducts of another pathway. Either way, these papers raise some interesting possibilties, and pretty convincingly show TCR sequence popping up where it's not supposed to, so they've got a welcome entry in this series.

The next (and final) entry into this week of TCR blogging brings us up to the present crop of non-T-cell TCRs, including the papers that actually set off my interest in the topic. Having presented some of the papers to my own journal club I won't be surprised by a mixed response; who wouldn't want to double check the presence of TCRs in no fewer than three other non-lymphoid white blood cell types?

On to part 5, where we finish exploring the landscape of where TCRs emerge

T-Cell Receptors, Part Three: TCRs Where They Shoudn't Be

Back to Part 2: A History of TCR Discovery

Wait, this isn't the right lymphocyte!

As the name implies, TCRs are receptors that you find on T cells. They're pretty well named in that regard. Seek ye TCRs, find ye T cells. Or so the story goes.

Biology is a busy, messy place, and our cells and the proteins inside them don't always behave as the textbooks might want them too. This series started out by me wanting to share some of the interesting stories I've stumbled across in my research, of TCRs popping up were perhaps we might not have expected them...

The first hints we have that perhaps the 'TC' in TCR isn't 100% accurate came not longer after the discovery of the delta chain, and in a very similar cell type. Several papers in a row show transcription of truncated, non-rearranged TCR gene transcription in B-cells, both alpha and beta chains, in normal and cancerous B-cell lines.

This perhaps isn't that surprising. B-cells are the other branch of the adaptive immune system that also produces variable antigen receptors (via V(D)J recombination). They're closely related lymphocytes, with related functions, that share an ancestry in the common lymphoid progenitor cell.

What's more, due to the shared developmental origin, somatic recombination and related purposes of the TCR and BCR loci, it's possible (if not likely) the various loci would share certain aspects of transcriptional control - the recombination machinery is certainly conserved among them.

Bearing in mind that the individual gene segments have their own promoters, and are capable of individual, non-rearranged transcription (as per the accessibility model of V(D)J recombination, where such transcription is required), it's not a huge leap to imagine that maybe B-cells just have some factors left over that drive a bit of TCR expression, of little physiological significance. The fact this story sputtered out not long after its inception might lend weight to that possibility.

The odd lymphocyte out?

I should probably mention that black sheep of the lymphocyte family, the TCR-bearing NKT cell. Now we're talking proper TCR here; rearranged genes, functional αβ heterodimers signalling on the cell surface, the whole shebang.

Discovered in 1987, these chimeric cells typically express a semi-invariant TCR, although there is a rarer, less studied type that expresses a more variable repertoire.

However, NKTs don't really have a place in this story; they're really just T-cells that went to NK cell school, so it's no surprise at all they have TCRs.

From here on in we're in much murkier territory.

The prostate examination

A little time passes before we find TCR expression popping up in new places. In 1999, while searching for highly expressed prostate genes, NIH researchers - headed by Ira Pastan - noticed an odd transcript popping up; none other than the TCRγ gene.

Oddly, it looked like the γ chain going solo; there was no δ, nor CD3, which you would have expected if the transcript was being carried in by infiltrating γδ T-cells. In situ hybridisation experiments revealed that the TCRγ transcript was being produced by acinar epithial cells.

A closer look at the transcript itself revealed that it wasn't even a full, recombined receptor gene being expressed. Instead, the sequence started with a particular TRGJ region*, which was correctly spliced onto the γ constant region.

Having done some in vitro translation on these odd γJC transcripts, they knew what size proteins they might make, if they were indeed translated in vivo. Well, fast-forward a few years and the same group is back with a vengeance. I mean antibodies, they're back with antibodies.

In their follow up paper, they show that the smaller of the two proteins encoded by the transcript (in an alternate reading frame to the typical γ transcripts we know and love) is not only expressed in the prostate, but in prostate cancer, and several breast cancer cell lines to boot. In welcoming it to the world of the translated, this protein gets a name; TCRγ alternate reading frame protein (TARP).

Over the coming few years, TARP enjoys a small but regular input of papers, showing that while involved in regulating growth, it itself is up-regulated by testosterone, via an androgen-receptor binding site in its promoter, and ends up expressed on the mitochondria.

This is pretty interesting stuff; we have (part of!) an immune receptor, being expressed in epithelial cells, under different regulation, with a different subcellular location, for presumably different purposes, but all off the same locus (which is itself within/part of the locus for another antigen receptor chain).

However, there is a whole other story that's come to dominate the TARP field in recent years; that of cancer therapeutics.

Being expressed seemingly only in healthy prostate tissues, or prostate and breast cancers, TARP makes for a pretty attractive drug target. 

One possibility is to use portions of the TARP promoter to design gene therapy vectors that will be specifically expressed in prostate tissues, allowing you to throw in a for something that (for example) inhibits cancer progression.

Closer to my own heart is the burgeoning field of TARP immunotherapeutics. Two groups have isolated CD8+ cytotoxic T-cells (CTLs) targetted against TARP peptides (HLA-A2 restricted), while another group has found some TARP-specific CD4+ T helper cells. The idea is this information could be used to possbily generate anti-TARP cancer vaccines, or T-cell therapies against established tumours.

What's more, one of the TCRs from TARP-specific CTLs has been engineered back into peripheral blood T-cells; T-cells with this TCR genetically engineered (or transduced) into them were shown to be active against HLA-A2+, TARP+ breast and prostate cancer cells, which could make for a promising treatment.

Of all the cases of wandering-TCR we see, TARP is perhaps the best appreciated and studied (I guess being a possible cure for cancer will do that). In the next two instalments in the series, we'll see some cases where the evidence is a little slimmer, or the findings perhaps a little bit more controversial, but hopefully still just as interesting.

*A note on TCR gene segment nomeclature (as defined by IMGT, whose system I use throughout my blog, along with their numbering). The first two characters represent the gene type (TR for TCR, IG for immunoglobulin), the next letter is the chain (alpha, beta, gamma, delta), and the final letter is the type of region (V, D, J or C).

On to Part 4, where we almost see TCRs where they shouldn't be