Genetic Strategies Used by the Immune System
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Lindau Nobel Laureate Meetings204 / 340
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Meeting/Interview
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Computer animation
Transcript: English(auto-generated)
00:22
Thank you, Professor Holm, for the nice introduction. I also wish to thank Count and Countess Bernadotte for the invitation to this wonderful conference. I and my family are enjoying our stay in Lindau enormously.
00:43
And I'm particularly pleased to have the opportunity to meet and chat with bright, enthusiastic, and younger scientists here assembled, from whom I sincerely hope that some Nobel laureates will be produced in the future.
01:03
The subject of my talk is a genetic strategy utilized for immune recognition. And I would also like to tell you briefly about the new T cell subset, which we accidentally
01:21
discovered a few years ago. Now, as many of you know, the most critical event in mounting immune response is a recognition of foreign objects, collectively called antigens. The molecule entrusted with this task are two sets of glycoproteins called antibodies,
01:43
or immunoglobulins, and T cell receptors, whose most intriguing feature or property is their variability in structure. In contrast to all other known proteins,
02:00
these recognition proteins of the immune systems come in millions, or perhaps billions, of slightly different forms. The difference enables each recognition molecule to recognize a specific antigenic determinant. And I would like to start the slide.
02:23
OK, the lymphocytes which produce these recognition molecules consist of a large number of clones. And the member cells of each clone, which are illustrated here in a very abbreviated fashion,
02:41
express antibody molecules with a particular structure as cell surface receptors, as indicated here. When the antigen enters the body, which is indicated by this blip here, its chemical components, antigen's chemical components, are
03:01
screened by this diverse array of lymphocyte clones via their cell surface antibody molecules. And a small number of lymphocytes that happen to bear antibody molecules with a sufficient affinity for the antigenic determinant in question interact with the antigen.
03:24
This interaction then selectively triggers. And activate these small number of lymphocyte clones, represented by clone number three, for specific propagation and for eventual differentiation
03:42
to plasma cells, which actively secrete the antibodies directed to that particular antigen. So this is the essence of Barnett's coronal selection theory, which is now a fact established by many evidence.
04:02
Therefore, the immune system has adopted what one might call a shotgun approach, or shotgun strategy. In other words, prior to the arrival of the antigen, or enemy, and without even knowing what kind of enemy
04:21
it is facing, our body builds up its defense by producing a vast array of weapons that can deal with almost any enemies, many of which, in fact, never arrive in our lifetime. Well, it sounds like a familiar story
04:41
for building defense in the real world. Well, at a glance, this sounds like an awfully wasteful and, one may say, not very smart approach to deal with our adversaries. However, in the biological world,
05:00
it turned out this is probably the only way the vertebrates can survive in the hostile microbial environments. Because of the relatively short generation time, microorganisms can generate variants or mutants very quickly, and the nature of these variants
05:21
are often unpredictable. Without a shotgun approach, the recognition machinery of the immune system will be quickly exhausted and the vertebrate host overpowered by pathogenic microorganisms. For the shotgun strategy to be effective,
05:42
one can easily imagine that the repertoire of the antibody that a single organism produces must be very large. We cannot determine precisely how large this repertoire will be,
06:00
but it has to be large enough to include all those antibodies that would interact with microbial variants and the mutants that may be generated even in the future. Indeed, our immune system can respond to entirely man-made antigens
06:23
that never existed on this earth. So one can imagine how large the antibody repertoire has to be. This, however, poses a great dilemma because antibodies are, after all, proteins, and different proteins need different genes,
06:43
and yet our body cells have a limit in the number of genes they carry, which is no more than 50 to 100,000. Even if as much as 10% of these genes deserved to encode just antibody molecules,
07:02
which is highly unlikely possibility, that would amount to mere five to 10,000 antibody genes. Now, can we homo sapiens deal with a rapidly changing microbial environment
07:21
with just this few antibody genes for millions of millions of years? Now, as it turned out, we do not, in fact, inherit a single, complete gene for this set of proteins for antibodies from our parents. Instead, we receive a limited number of gene segments
07:45
encoding different parts of antibody polypeptide chains and assemble these gene segments in a variety of fashions in our individual lifetime. And next slide give you cartoon
08:03
of the arranged organization of these gene segments, which are inherited not by us, by a mouse, from his or her parents. And what is shown here on the upper part of the slide,
08:20
the gene segments before the assembly process, they come in three different forms, which are designated as V segment, D segments, and the J segments. Each of these gene segment pool contain multiple copies, which are different from each other.
08:41
And in the cell in which antibody is produced, B lymphocytes, these gene segments assembled in a random fashion as indicated here as one example. And this can now encode the entire polypeptide chains.
09:01
And it is important to realize that these V, D, and the J segments are all involved in encoding the amino acid residues that play a role in the formation of antigen-combining sites on the antibody molecules. This is a bad-view picture
09:22
of the typical antibody-combining sites, and these bright colors indicate the residues provided by these three different gene segments that are involved in the formation of antigen-combining sites. So, antibody genes made up with different combinations
09:44
of gene segments encode antibody molecules with different antigen specificities. Now, one can calculate what will be the potential combinations one can generate from known or estimated number of gene segments
10:02
that you inherit. And this is done in the next slide for a mouse again, and a human will have a similar situation. So, HC means a heavy chain, LC is a light chain, light chain, so-called variable region, is encoded by only two types of gene segments
10:22
called V and J, unlike heavy chain gene variable region, which is encoded, as I said, with D and J. And these are the copy numbers of each of these gene segments that a single organism has, and in some cases, the precise number has been determined,
10:41
and in other cases, these are the pretty good estimates. So, you multiply these numbers to get the total potential combinations, which is 24,000 in mouse heavy chains, and 1,200 for light chains. Now, we know that both heavy and light chains can contribute in the formation of specificity,
11:02
antigen specificity. So, now you multiply these two numbers, and you come up with 30 million different combinations, starting with just about 600 different gene segments that you inherit, or mouse inherit. Now, in addition to this combination
11:20
of different gene segments, which helps enormously in amplifying the specificity, the diversity of antibody molecules, it turned out that in the junctions between these gene segments, VD and the DJ, there exist deletions and insertions of nucleotides
11:42
of undetermined, unpre-determined lengths, and that obviously generate extra diversity in the junctional regions. So, it turned out these molecules in the junctional regions are so-called hotspots, where the diversity is particularly high.
12:03
However, this is not the only source of so-called somatic origin of antibody diversity. It turned out there is second major mechanisms which will generate further diversity among this set of proteins, and that is the somatic mutations, hyper-mutations,
12:24
high rate of mutations that occur, specifically in the assembled immunoglobulin genes so-called V regions, that takes place during the B cell development. And these are three examples where each of these dots
12:41
represent the position of base changes, single base changes, that has been identified precisely by determining the nucleotide sequence of these genes before and after gene segment assembly. So, next slide summarizes all these sources, origins,
13:01
or mechanism that generate and diversify the immunoglobulin or antibody genes that are inherited from the parents. So, as I already mentioned, the somatic recombination, or assembly, of these gene segments can be divided into three types.
13:21
The random joining of VJ or VDJ gene segments, and then imprecise joining ends, which generate a deletion at the joints, and also template independent insertion of nucleotide at the VD and the DJ joints of heavy chains. In addition, there is a somatic hyper-mutation mechanism,
13:43
and thirdly, the heavy and the light chains can assemble with some degree of randomness and generate different combining sites. So, putting all these together, starting with less than 10 to the three, 1,000 inherited gene segments,
14:01
the organism like mouse or human can potentially generate over 10 to the nine specificities in order to cope with the enormously large antigenic environment. Now, one may remember that evolution is rather conservative.
14:21
In other words, they don't create something unless there is a good reason. Now, one wonders that these two extraordinary mechanism, the mutation and the recombination, occurring at the very high rate in the lifetime of individual,
14:40
why did the organism, or did we, have to resort to two extraordinary genetic mechanism to carry out the task of diversifying the antibody molecules? And this was a little bit mysterious for a while, but now, as the mechanism regulation of these genetic events being studied,
15:02
we have a pretty good idea of how this happened. And to put it very in short, these two genetic mechanism, recombination and mutations have sort of division of labors. They both fulfill the purpose of diversifying
15:21
the gene product, antibody molecules, but they do slightly different things. And in order for me to explain to you, I want to show you a slide, again, the cartoon which was taken from a textbook. And this slide shows the cause of the typical immune response
15:42
when the animal is immunized with particular antigen, and then the antibody concentration in the serum is fold against the time, against the days. You usually see this kind of kinetics of changes, of the titers, the antibody titers.
16:03
So this is a so-called primary response, and this is a secondary response. And there are three important differences in the primary response and the secondary response. One is obviously the extent of the response is much greater in the secondary response.
16:20
And the secondary response is much quicker in the second time. And thirdly, the point that is relevant to the discussion I want to make is the fact that the secondary antibody usually have a much higher affinity than the primary antibodies.
16:40
And it turned out this difference has very much to do with the regulation of these two genetic mechanism, as indicated in the next slide. So this is a drawing, a cartoon, similar to the one I showed you earlier for the coronal selection, except more details now incorporated.
17:02
So here is the resting B lymphocyte expressing the antibody receptor on the surface. So by this time, all assembly of gene segments have been completed. They wait for the arrival of antigens, which are indicated by these dots here. And then as I indicated earlier,
17:21
the specific subpopulation of these clones will be expanded as shown in here and generate the plasma cells by primary immunization. And it turned out during this process, no or almost no somatic mutations occur.
17:41
So the most of the primary antibodies, primary response antibodies have no mutations. And because these antibodies are made using blocks of DNA segments, blocks of DNA which have gone through the evolution, one cannot really custom make
18:01
the high affinity antibodies to a specific antigen, although they have sufficient affinity to it and therefore cells bearing those antibodies are activated, they don't have very high affinity. Now during this primary immune response, there is another pathways of B cell development, which is the formation of so-called memory cells.
18:22
And it is during this process of the formation of the memory B cells, the somatic hyper mutation machinery is activated. So the nucleotides are altered more or less randomly during this process and only those small mutants of B cells
18:40
which happen to have antibody receptors whose affinity to the specific antigen that we are talking about is improved, higher affinity. And as a result of that, the secondary antibody has much higher affinity. And you can look at this as a sort of two-wheel mechanism.
19:04
So this is a coarse tuning by assembling blocks of DNAs which went through the evolution to generate the primary antibodies with low affinity. And then once antigen is fixed, then we know how to improve the affinity
19:20
by letting antibody mutates randomly and letting antigen select those small fraction of cells which happen to have the antibody with much the higher specificity. So this is a coarse, you can say this is a coarse tuning and this is a finer tuning for specificity.
19:44
Now I would like to turn to related problems, namely the recognition of antigens by T lymphocytes. As most of you know, that immune systems are composed of or contains two major type of lymphocytes,
20:03
B lymphocytes producing antibodies and the T lymphocytes which produce T cell receptors for the recognition of antigens. Now it turned out that the T and the B cell recognize antigens in quite distinct fashion. I have already mentioned about how the antibody
20:23
can be a receptor on the surface of B cells as well as freely circulating molecules. In either case, the antigen can directly bind to these antigen combining sites on the antibody. In the case of T cells, the recognition molecule
20:41
is strictly on the surface of T cells as a receptor and it is called a TCR, T cell receptors. Now what they recognize, what they specialize in the recognition is the antigens presented on the variety of cell, on the surface of variety of cells which can be collectively called
21:03
antigen presenting cells, APC. It turned out when, for instance, our cells are infected with viruses, virus proteins which should act as antigens processed into peptides, small peptides in the cell, within the cell and these peptides bind
21:22
to a specific set of proteins, like proteins encoded by major histocompatibility complex, MHC class one or class two molecules. And this complex between the antigen-derived peptide and peptide-presenting MHC molecules
21:41
collectively recognized by T cell receptors. And as a consequence, the T cell can, for instance, kill the virus-infected cell by its own ability to raise the target cells after this recognition takes place. Now for some time, the biochemical,
22:02
the basis of this T cell receptor was difficult to combine. And the study on this antigen-presenting molecule proceeded earlier and as shown in the next slide, few years ago, Don Wiley, Jack Strominger
22:20
and their coworkers at Harvard University reported extremely beautiful work on the X-ray crystallographic analysis of human MHC molecule. This is not a T cell receptor. It's a peptide-presenting molecule, MHC molecules. And as you can see here, this blue part is again
22:43
the bird view of the peptide-presenting area of the MHC molecule and this pink part is supposed to be a known peptide. Now for the structure of the T cell receptors,
23:01
in 1984, the molecular geneticist succeeded in cloning these genes using a very special method called the subtractive cDNA cloning. And as some of us expected, these genes encoding T cell receptors,
23:21
similar to immunoglobulin genes but distinct from them and they use a same type of strategy of somatic assembly of gene segment. This is the alpha locus and this is the beta locus. And as shown in the next slide, most T cell receptors are heterodimers
23:41
composed of alpha chain and the beta chain. And this, the membrane distal domains of these molecules contains the residues which are involved in the binding to peptide and MHC molecules. Now I will not talk very much
24:00
about this conventional T cell receptors. Instead, I would like to talk about what is displayed on the right half of this slide. During the search for the genes coding for alpha and beta T cell receptor genes, we run into accidentally a third gene which we named the gamma genes
24:22
because it was a third one, which showed the number of similar properties, properties similar to the genes encoding alpha beta chains. And in other words, these genes can rearrange dramatically in some T cell subsets. They have some significant sequence homology
24:41
to alpha beta genes. Now there was nothing was known about the possible product of this gene when the gene was discovered. It was so that all T cell receptors are made of alpha beta heterodimer. Therefore, the research in the field has progressed
25:01
and still progressing in a diverse fashion. Namely, gene was discovered with no knowledge of gene product or cells expressing those genes and suddenly nothing was known about their function. Now subsequently, the first gene was discovered
25:21
by Davis Chen at Stanford University and it was later shown that these two genes, gamma and delta, form a heterodimer to encode a new T cell subset, new T cell receptor subset. Now if you look at the conventional lymphoid organs,
25:43
peripheral lymphoid organs where T cells are abundant, which is the spleen, the lymph node and the peripheral blood lymphocytes, it turned out almost all T cells in these organs are of alpha beta type. No more than 3% of the T cell in these organs
26:02
bear gamma-delta T cell receptors. So one wonders whether this is simply a minor component of T cells, may be dispensable for the survival of animals, but later studies by many people,
26:21
including ourselves, showed that these T cells actually are distributed in the area of the body which were totally unexpected. Namely, in the epsilium layers. For instance, epidermal tissues have many T cells, associated T cells, which express
26:41
this particular type of receptors. And later it was shown that the gut epsilium cells also have these T cells preferentially and the lung epsilium cells do. The tongue, if it's a female, uterus and the vagina epsilium layers
27:00
also have selectively the gamma-delta T cells. Now you might notice that these are the part of the body which are exposed to outer space. Some are inner side of the body, but they face outer space. So it was postulated that these gamma-delta cells
27:22
have evolved to protect the surface of our body, which is a primary entry site for some selected pathogens. Now this slide simply shows example of the identification of gamma-delta T cells in these epsilium layers.
27:41
In this case, it's epidermal tissues. This is so-called immunohistology of the section of the skin, and this is staining by anti-gamma-delta antibody, indicating all these black-dark areas indicate the existence of gamma-delta cells. This is a control experiment which was carried out
28:02
using antibody against alpha-beta T cell receptor, and as you can see here, alpha-beta cells are scanned in this part of the body. Now another interesting feature or important feature of the recognition by T cells is what I already mentioned, namely the involvement of the MHC-encoded proteins
28:25
as a presenter of antigens. And as I said, it's been established for the case of alpha-beta T cells, the specific set of MHC molecules called the class one and the class two, which are mapped in the left part
28:41
of the large major histocompatibility complex encode these molecules. Now our recent studies and also studies by others indicate that these T cells, gamma-delta T cells, recognize not these classical MHC molecules,
29:02
but the new type of MHC molecules which had been crowned in the past, but their function had been unknown. These genes are called TL genes, and there is a structural similarity to the classical MHC molecules, but they are distinct proteins.
29:20
So the next slide is a model of T cell recognition by the recognition of antigens or MHC by gamma-delta T cells in contrast to the recognition of antigen presented by classical class one or class two molecules by alpha-beta T cells.
29:42
Now as you notice, we still do not know the exact function of this new subset of T cells. However, during the past year or so, many people got interested in understanding the role of these T cells in the immune responses,
30:01
and as a result of these studies, we have a large number of patients, human patients, in which the possibility of involvement of the role of gamma-delta cells have been implicated. So I just want to show you the list of,
30:21
I don't know if you can see this slide well. This is the list of diseases in which the possible involvement of gamma-delta T cells has been suggested. For instance, at the top, ataxia, telangiectasia patients have a defect in the DNA repair
30:42
and apparently in DNA rearrangement involving large DNA segments, because we and the J gene segments are much more apart in the T cell receptor alpha-beta genes than in gamma and delta genes. Alpha-beta T cell populations are more affected in these patients than gamma-delta T cell populations.
31:04
So we will see whether this alteration in drastic alteration in the ratio of the alpha-beta and the gamma-delta cell have anything to do with this particular disease. Now, another interesting series of cases is the finding that increased number
31:22
of circulating gamma-delta T cells have been found in certain immunodeficiency diseases and in immunodeficiency generated after bone marrow transplantation, as indicated there. Yet another interesting series of cases is the autoimmune diseases,
31:41
such as in rheumatoid arthritis and various other diseases which are sought to involve a variant immune responses, as well as infections, in particular infections by micro-bacteria. Gamma-delta cells have been found in increased numbers in the blood
32:01
and in locally infiltrating tissue regions. Finally, the tumor-specific gamma-delta cells have been found in patients with B cell, some B cell lymphomas, and with acute lymphoblastic leukemia. Such tumor-specific gamma-delta cells can be grown in vitro,
32:21
since they raise autologous tumor cells very efficiently. These cells might be used in the future. Instead of so-called lac cells, lymphocyanin-activated killer cells for tumor therapy. We will see whether the gamma-delta cells
32:40
really play a role in these disease conditions and whether we can develop some therapeutic method using these T cells. Now, I would like to summarize what I have told you. We have shown that in the immune system, both mutation and recombination, which are normally reserved
33:01
as events occurring in evolution, utilized during the development of organism to cope with fast-changing world of pathogenic microorganisms. While thus far, this is the only known system in which these genetic changes are exploited
33:22
during development for the benefit of higher organisms, one wonders whether it is indeed unique to the immune system. This only the future study will answer this question, and I find this is an intriguing question.
33:40
Now, in addition, our molecular genetic study of T cell receptors led to the discovery of entirely new T cell subsets, which seem to play a distinct role in the immune system. Thank you very much.