Welcome back to Immunology with Numbers and apologies for a long break. Struggling to interpret high throughput T cell receptor sequence data from antigen stimulated repertoires prompted the following thoughts. As always, I would love to receive comments and especially criticism. Also, do take a moment to have a look at our new Chain/Noursadeghi web site.
The vertebrate immune system uses a combination of somatic gene rearrangements and non-template junctional editing to produce a very large number of different receptors which bind a wide variety of antigens. Recent T cell receptor and B cell receptor high throughput sequencing have allowed some preliminary estimates of the size of the potential diversity . The most thoroughly analysed datasets are those from the T cell receptor β chain. The consensus appears to be that the number of possible different human β chain sequences is in the order of 1014 or above. The α chain diversity has been studied much less. Nevertheless our own sequencing, as well as other published studies suggest that the diversity in the human α chain is in the same order of magnitude. We don’t know the rules which govern the interaction of α and β chains, or what restrictions on such pairings exist. But, even if we assume that such rulings impose a significant restriction on the total number of possible receptors, the number of possible receptors is likely to exceed 1020. And the overall germ line diversity of antibody is likely to be of the same order of magnitude.
It is immediately apparent that these large numbers pose some interesting conceptual problems in terms of specificity. If, as an extreme example, only one single receptor (defined by a unique germline sequence for both chains) recognizes a single antigen epitope, then since an individual has only around 1012 lymphocytes, almost all individuals would be missing the relevant receptor from their repertoire and would never recognise the antigen in question. Even if an individual did have such a receptor, it would at best occur only once during recombination. The frequency of individual T cell clones can be extremely low, possibly as little as 109. Despite the relatively high motility of T cells, dendritic cells sample T cells at rates of less than 1000 cells per hour. Each dendritic cell presenting the specified antigen would therefore have to wait many hours before encountering even one relevant T cell. What might be a solution to this paradox arising from the sheer number of possible receptors ? One possibility has been discussed in some depth by Alexandra Walczak and Thierry Mora. As they and others have shown, recombination is a random but non-uniform process. Some receptors are produced at much higher frequencies than others, and indeed are produced so frequently that they are apparently expressed in all individuals (so called public clones). In their most recent publication they suggest that recombination machinery, as well as the germline sequences themselves, have somehow evolved to produce receptors which are naturally selected and perhaps therefore more useful than others. This idea could be extended to suggest that the commoner receptors are those which have evolved to recognise common pathogen components : a sort of adaptive pattern recognition receptor.Putting aside the difficulty of envisaging a mechanism by which an apparently stochastic process which generates non-germline encoded diversity has “evolved” to favour certain sequences over others, this answer can at best account for only a small proportion of the diverse array of antigens against which the human immune system responds.
I would like to suggest that the diversity paradox can be resolved in only two inter-related but distinct ways. One approach is to increase receptor cross-reactivity at the expense of loss of specificity. If, for example, a single antigen epitope is recognised by 1010 different receptors, which are each generated with a wide range of probabilities, then even from a pool of 1020 possible receptors, each individual will have a high probability of having at least a few receptors which recognise any antigen, and the frequency of available receptors within an individual will in general be high enough that a new immune response can be generated within a few hours. The cross reactivity of the T cell receptor has been debated for some considerable time. A recent elegant paper from Christopher Garcia and Mark Davis’s laboratory demonstrate clearly that a single T cell receptor can indeed recognise millions, or even thousands of millions of different peptide targets, although interestingly the targets tend to share conserved “hot-spots” which represent conserved areas of TCR/peptide interaction. Of course, the correlate of high cross-reaction is low affinity, a well known property of T cell receptors, which is offset by the extreme sensitivity of the signalling mechanism, and the avidity factor which arises from displaying many identical receptors on the cell surface. The most common TCRs generated by the recombination machinery are likely to be of lower affinity than rarer higher affinity TCRs, but they may provide some cover while the higher affinity clones have time to find antigen and expand. Nevertheless, the overall characteristic of this highly diverse TCR repertoire will be low affinity and high cross reactivity.
The second approach is provided by somatic hypermutation, the characteristic feature of B cells and antibody. In this case, once again low precursor frequencies can be mitigated by having early responses mediated by low affinity, cross-reactive clones. This ensures that sufficient numbers of precursor cells exist for any epitope and can be recruited into an ongoing immune response within hours of exposure to an antigen. However, once these clones have been selected and expanded, somatic hypermutation provides a way that the BCR can be gradually fashioned to provide higher and higher affinity by repeated rounds of selection. Since selection only has to operate on the initial limited pool of low-affinity precursors recruited into the immune response, the initial enormous diversity of the BCR will not preclude the gradual emergence of antibodies with extremely high specificity, and very low cross-reactivity.
In conclusion, the extraordinary hypervariability built into the immune system’s receptor generating system imposes severe constraints on the ability of an individual to recognise anything at all. The evolutionary solution is to combine two sets of lymphocytes, T cells which respond at low affinity with extensive cross-reactivity and B cells which evolve gradually to produce antibody of high affinity and low cross-reactivity.