How to Measure Immunological Specificity
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Transcript: English(auto-generated)
00:14
Good morning, and thanks for coming and a warm welcome to you
00:21
to this Agora talk and Our Agora speaker is one person and that's Rolf Zinkernagel Well known of course, so he doesn't really need an introduction I would call him an immunologist and he's interested in immunology
00:41
and as you may know from the beginning on and I count immunology since 1901 when the first Nobel Prize in medicine was ever given to a German Physician and at that time the term immunology wasn't that frequent that was Emil Biering and he showed that the real ingredient of
01:01
immunology is specificity. Now immunology was at that time mostly defined with by antibodies and antibodies were specific for the antigen and that was the second Nobel Prize in my opinion in immunology to Paul Ehrlich in 1905 and Everybody talked about antibodies at that time and he talked or she talked about
01:23
immunology until later in the 60s 70s It was realized that there's a second arm in immunology And that's the T lymphocytes and that is what Rolf together with Peter Doherty Worked on and they showed then that T cells are much more complex in what they see
01:42
They see an antigen in a reference structure, which is the major is the compatibility complex molecule and that's what they got their Nobel Prize for before the discoveries concern the specificity of the cell mediated and Now comes immune defense and I would say that is something that Rolf always was interested in not just
02:01
immunology but immunology of infectious diseases and Immunology got bigger and bigger in the kind of area They were interested in tolerance memory, but specificity Was always in the center now Rolf is Swiss. He was born there. He studied there
02:22
He got his first positions there. Then he went to Australia where he met Peter Doherty and together as I said before they worked on the On these antigen specificity or the specificity of cellular immunity They received the Nobel Prize. He went to the US but then back to Switzerland at the ATH where he still is active as an emeritus
02:46
Professor and I think I've talked enough everybody knows him anyway So all of the stage is yours to talk about measurement of immunologic specificity Thanks very much Stefan As usually in science and his talk about half is right we don't know which half right so
03:08
Let me talk about Specificity because as just introduced specificity is You know is the base of how to measure all other phenomena, so it's extremely important
03:22
You know what we mean by specificity and of course we have known that for a long time because so-called serotypes of infectious agents that kill us like Poliovirus type 1 type 2 or 3 Tell you what a serotype is if you are immune against Polio 1 you are not immune against Polio 2 and 3
03:46
very simple But there are thousands of antigens that are immunologically, you know via antibodies shared between Polio 1, 2 and 3, but the protective antibodies are
04:01
exclusive for the serotype Now infections kill you before You get sexually mature that's the key point because at the end of the day we always only have to Get up to 22 years or something like this because then we have our kids with 12 or 14
04:23
We can rise them and then disappear from the evolutionary scene because we are not needed any longer You cannot really select for wisdom Because wisdom usually comes too late so That of course has a problem if you interfere because the longevity
04:45
Creates the biggest world problem we have at the moment too many people So we are all a bit schizoid Schizophrenic in that we think it's very important to continue improving longevity But on the other hand we all realize
05:03
That this causes problems. So this is the basis Let's see what oh here we are so specificity We have to understand that and this then translated into understanding of memory or so-called immunological tolerance that is
05:22
this so-called distinction between self and a infectious agent Now the first problem the immune system has to face is the size of the agent You also could say the size of the antigen
05:41
Because a lymphocyte is about five to seven Micrometers and that's about the size of a staff or a candida But many important infectious agents are much bigger than macrophages or or granulocytes So there's a problem
06:02
How does a system that basic basically is made out of cells of this size You know to deal with these huge joints and of course, there's only one solution Things have to be degraded Digested and that's why these things are much more difficult to control immunologically
06:22
Than things that are within the size of a lymphocyte particularly the viruses and in fact viruses are about The optimal target for both B cells to make antibodies neutralizing antibodies Of course and size a toxic T cells
06:41
Now How big is the immunological target if you look at Immunology in the literature 99% of Immunological studies on antibodies is done with so-called hapten
07:02
that's phenolic groups that are slightly modified in addition and This is basically a six carbon type of ring and the size Translated into real antibody binding site and a viral target size of
07:25
A hapten is just very small and therefore no surprise antibodies against so-called happens have a lousy affinity and Their frequency is enormous. It's about one in fifty to one in a hundred
07:44
But we all know that Neutralizing antibody producing B cells are very rare ten to the minus five to ten to the minus six And their affinity or binding qualities at the level of ten to the minus ten Ten to the minus nine is the lower level
08:02
So there are huge differences how things are measured and there's no surprise that if you use this as your model Situation you probably get some answers, right? But most of the answers will be wrong because In the these high B cell frequency situation your limiting factor is not the B cell
08:26
It now becomes a T helper cells that help to switch IgM to IgG responses In this case in the neutralizing antibodies case that beer B cells are so rare
08:42
That the only limiting factor is the B cell frequency and not T help And that's of course the contrary what you read in your textbooks So Let me summarize we have serotypes of protective definition of a
09:03
specificity versus ELISA definitions the binding qualities differ by 10,000 or a hundred thousand and this of course has direct consequences in our judgment about the B cell or antibody repertoire
09:22
This indicates that the repertoire is about ten to the three And if you just think you know infectious disease like then we don't even have a thousand infectious diseases that Kill us easily or at least it's below 10,000
09:45
So the repertoire calculation of 10 to the 12 is theoretically Not completely wrong, but it's certainly not right either Then of course accessibility of antigen plays a role
10:01
you know, for example, if you make a negative staining of a antibody coated Enveloped virus, this is a rabies like virus you see that actually the neutralizing antibodies stick Strictly to the surface. They cannot squeeze in between the glycoproteins
10:20
There's simply no space so the construction of the virus actually makes or Makes for exclusivity and I think this is a general rule for for serotypes so Then comes of course the variability or escape type of
10:41
Genetic variability like for influenza or HIV or other viruses that simply escape immune responses But only viruses can do that that either in the individual are not Cytopathic that is don't kill you within 20 years HIV is such a case or
11:00
That don't kill the whole population because you know epidemics takes time and this is the case for influenza Now If you Disregard these general rules and this has happened repeat repetitively over the last 100 years
11:21
You know the more sophisticated Sophisticated Trickers think this cannot and must not be true and therefore there must be cross-reactive or cross-protective antibodies that Protect you not only against I take an extreme case polio 1 but also against polio 2 or 3
11:44
They don't use polio because the exp you know the the the facts are too clear But they take influenza or HIV and now they argue if I make antibodies against these Common determinants in the stalk like area of the virus
12:03
But not against the tips like in the case of these envelope viruses I may find antibodies that actually interfere somehow with the detection methods and In fact, you can find such antibodies and now comes the question
12:23
is the test reliable or not and you always use a indirect test because you can't do the definite test in viva in humans So what is being used in influenza for example is hematolutination?
12:44
Inhibition which is not a direct test, but it correlates reasonably well, but it's not the proof and I think that is a real problem. So let's now go back
13:01
Into a lab condition where we take mice on the so-called specific Pathogen-free conditions that is they live almost Completely free of any pathogens because remember if you get vaccinated, let's say with polio or with
13:21
cholera toxin or with measles Nobody can do a controlled experiment because the epidemiological circulating type of Infectious agents you cannot control against otherwise you would have to put the kid after vaccination into a plastic bubble
13:43
Otherwise, you know, you don't know is it so-called classical memory or is it? re-exposure and the re-exposure of course can also come from within or from without but this is Disregarded by those who think so-called immune protection via vaccination is
14:04
exclusively via a parallel of Neurological memory one scene always protected and I just illustrate that with a rabies like virus If you infect these controlled hygienic like controlled hosts and you look for
14:23
Neutralization you find that within 30 to 50 days The level of neutralizing high affinity antibodies has fallen below protective levels Whereas if you measure via an ELISA test
14:40
Here you measure at 10 to the minus 10 here. You measure at 10 to the minus 5 or 10 to the minus 6 You have titers, you know for a year in a mouse or two years in the old mouse So it depends on you know, which I say you choose To make your conclusion and of course if you think memory is important then you choose an ELISA
15:05
I say if you think it's protection and antigen plays a role you of course only Use protection and protection is very easy to measure at the end of the day legs are either up
15:21
That's bad or legs are down. That's good Now in T cells we have a similar problem The high-quality T cell Specificity measurements are the biological ones like for protection with antibodies
15:41
we can measure T cooperation with B cells to make IgG so-called T health or we can look for protection in vivo against tumors or non-cytopathic viruses And when you do that you usually get a very defined answer but
16:08
Less than 1% of immunologists use this type of readout most Do tetramer staining, you know where you have artificial types of reagents that have four
16:21
peptide MHC complexes or gamma interferon release From T cells that get laughed at or lysis of LPS blasts these are artificially blasted lymphocytes and those release
16:42
Stain or chromium or any other marker if you just stare in them because they are very very fragile and so on but never people do not use fibroblasts or Epithelial cells simply because They can't see anything
17:01
So it's the same problem like with Elisa's versus protection or neutralization for the case of T cell specificity So one of the conclusion of course is be as tough as you can be in your experimental life Choose something that is close
17:24
to Infection survival and Close to those times and periods in life where these mechanisms become Very decisive so specificity is defined either by serotype
17:44
with antibodies and interestingly enough these serotypic definitions have been mostly coined for viruses For carbohydrates on on bacteria, but the definition of
18:00
specificity and the correlation with affinity for example Has not been renewed in terms of studies with bacterial toxins Because people think We know everything but that for example with diphtheria toxin The last really decisive studies were done in the 50s or 60s when many many methods were not available
18:27
So serotype or CD8 let's say CTL escape of virus such as LCMV in our case or HIV in humans and
18:41
this all really negates The existence of so-called broadly neutralizing or broadly protective Antibodies particularly in those cases where the where the agents are defined serotypically And of course this has a direct influence on our understanding of memory
19:02
Because if as I said if you use ELISA your memories forever if you use protection things fall apart Pretty quickly unless the agent persists in you or you re-encounter the agent and one classical example is of course cholera if a Bangladeshi
19:25
emigrates from Bangladesh to London to do a postdoc He was immune because of continuously exposed to the bacilli He loses that in London within a year. He comes back. He starts again with diarrhea
19:41
so That's not what we call Memory, and this is to in You know the cholera type of example is truth for most of the mucosal type of infections And of course tolerance suffers from the same technical aspect
20:02
So the conclusion is It's good to be an MD Because you can distinguish between legs up and leg down and if legs are up You know something important has happened And I think that's the best motivation to look into the details of the process
20:24
This of course is a very general state wrong promises Including these cross protective or cross reactive antibodies is simply wrong. I mean it doesn't help and
20:41
More general to measure something doesn't prove Biological relevance and I stop here Thank you, thank you Rolf this was
21:01
Precise and crispy and gives us room for discussions So the floor is open and we have the first comment or discussion. Can you get the microphone? Apparently we only have one person that poor lady has to run quite a bit So if I see hands up on the one side where she trust ends I will prefer those and then go to the other side. So don't take it personal
21:25
Very nice talk. I have a question concerning tolerance What about partial immuno tolerance? Let's say involving mainly B cells or T cells or both and or Not relating to macrophages Well the issue with so-called tolerance, you know tolerance as a term is an idea
21:49
So what is are the facts what we measure is actually no response. That's what we can measure Nothing else. We cannot measure tolerance
22:00
So no response can have several reasons one is like against all our blood proteins and blood elements The T cells Get so-called negatively selected or deleted That's the classical textbook definition but for example against a antigen that is
22:25
exclusively expressed in the periphery like on a fibroblast in your big toe or their tolerance does not exist and the example of that is we made a mouse Where we put a viral antigen
22:42
Under the rat insulin promoter to have that antigen expressed exclusively in the islet cells. So you have a mouse With islet cells that don't express all the islets cell and you but in addition a viral antigen and You wait for two years that mouse is happy and doesn't make a diabetes
23:04
But now you take a young man six weeks eight weeks ten weeks infected with the same virus from which you have taken that Antigen that viral antigen within eight days. All the islands are gone via cytotoxic T cells so the mouse
23:20
Doesn't make a response on the physiological conditions But if you bring the same as an agent into the lymphatic system Then hell breaks loose and I think most self antigens are actually of that second type Which of course is Optimistic for the tumor, you know therapist because most tumors are of the
23:47
Islet cell or big toe type that is you have something in the periphery The system doesn't notice because it's too far away And now you try to make an immune response against that tumor
24:01
The costs will be that you will cause enormous amounts of immunopathology by destroying own Normal cells so your ears may fall off for example For the questions comments. There were a couple of crackers in it for in this talk for immunologist
24:21
So I'm awaiting some and while you said you ready to discuss every point Sure down there So good morning professor Very happy that you're here. I would like to discuss more about autoimmunity Because of course the tolerance is broken in autoimmunity, but then what is your insights about?
24:42
immunity well You know, I Come back to one of my initial statements if we survive till 20 or 22 The species is basically, okay so two things happen after 30 in general that's autoimmunity and
25:06
solid tumors So in a way biologically You know it happens, but it's not really Selectively important in terms of coevolution, but now come back to so-called autoimmunity You know you may or may not have heard
25:22
Hall 2000 Talk is is this afternoon Yeah, okay, so you know there are extremists amongst us who say If you know the infectious agent, and it kills you and kills you in seven days You call it an infection with lethal or disease-causing consequences and their immunity is a must otherwise
25:49
You don't survive There are agents That do not destroy the cells in which or with which they grow and they doesn't matter whether you make an immune response or not
26:01
in fact non Cell destroying viruses such as HIV or HCV or HPV or LCMV in the mouse They get transferred from the mother Who doesn't make an immune response otherwise she wouldn't be a carrier
26:20
And these viruses are transfused to the offspring and the offspring cannot make a net response because it doesn't have a functioning immune system at the beginning of life So that's the point in life where you don't have a cell mediated surveillance and therefore you avoid
26:41
immunopathology that is cell destruction via the immune response an Optimal co-evolution now the third example is if you don't realize there is an infectious agent You know 1960 hepatitis B, C, A, D
27:01
G viruses were not known so there was a picture clinical picture of autoimmune hepatitis and Then 7, 10, 15 years later all these viruses popped up and now it's called well It's a immunological consequence of a viral infection
27:21
so to spin that just one step further I Like to argue if you don't recognize the agent or you can't see it You call it autoimmunity very simple view
27:42
One more comment down there, and then we go to my left and your right Thank you professor for a wonderful talk. I am a clinical immunologist from India So I want to discuss or I want to briefly talk about the intricacies as far as immunology is concerned So if we go back in history at one point of time we were targeting IL-2 to treat diseases
28:05
Now in the course of over the last 10 to 20 years We have recognized that IL-2 is the main thing that regulates deregulatory cells so now we want to you know favor IL-2 so with this This is just an example so what I'm trying to say is this is there is so much of redundancy
28:20
There is so much of pleiotropy that is going on as far as the immunological aspects of disease are concerned and with F with effects that are Magnificent so they can you could ideally logically be targeting something for a particular effect But you might discover that something totally opposite has happened so as a young scientist or as a young clinician. How do we?
28:41
Take care of that how do we? You know synthesize it in a particular way so that we can reduce the Extent of what is unknown or we can you know take it into account and be less erroneous while we take up studies or research Thank you Let me answer in three points one is
29:02
Don't believe anything You read about in textbooks or papers That you haven't done yourself Because you know in the literature you can find anything you want to Make your point
29:20
That's why I liked you argue about memory. You know there's thousands of experiments so try to to prove the contrary because I think That's very important the second point You use you mentioned interleukins you know when I began
29:48
with immunology There was Supermaidens that contained something like macrophage activation factor But nobody had any clue what that was and then came I'll one
30:06
Obviously the first one to be discovered because it has number one And then I'll two second one and so on now. We are about as 57 or something I don't know it's not important, but still you know if you find under limiting
30:22
conditions something new it's always Important and interesting to know some of that will disappear again Because probably it's not important, but if it is important it will stand the the critique of time So my advice would be
30:44
You know and we talked about that yesterday at one of these Master classes I think the biggest problem to Survive in science clinical science or basic science is to survive frustration Because frustration is about ninety nine point nine nine percent
31:05
Maybe huh and the rest is glory you know, but you have to survive these phases of Frustration and the only way you can do that and this You know includes myself is to have a question
31:20
Which you think is the most important besides your family and so on is the most important question to solve And then I think you will survive and then of course you need luck So Thank you one down there, and then we come to you I
31:40
Just wanted to ask if you believe CMV based vaccines could help circumvent The memory loss that you mentioned with the brilliant example of cholera toxin Well you see CMV all herpes viruses basically have solved the problem a long time ago Because they infect you Under the cover of maternal antibodies, so you don't make a
32:02
Encephalitis and they hide somewhere in long epithelial cells or kidney epithelial cells and stay there and sort of play ping-pong With the immune system, that's all there is needed now we Tend to grow up at super hygiene conditions
32:21
Where some of these natural transmission routes that have no disease consequence are avoided you know polio was the first obvious example and That of course is a problem because now you should Vaccinate the few that haven't gone through mother's kiss and transfer of of the herpes viruses
32:45
so You know how far should we go I? Think epidemiologically and medically except for organ transplantation CMV is not a problem one more question down here
33:01
Can we move to the microphone here? You raise your hand that you're and then there was one last question down there Thank you Thank you, depends on the time. I was very intrigued by this Concept that you proposed about difference from what you measure between what you measure what's real and was wondering
33:21
Do you think that actually we substantially? Underestimate herd immunity in vaccinations, you know vaccination schedules very precise and they're based on what you measure Do you think that actually there might be something different going on? Well, your problem is a bit more complex I think one part of the problem is mass
33:44
Psychosis and population Perception of of the value of vaccines And I think that's The biggest problem, you know but You know, there's a very interesting example if you look at measles virus
34:04
People have done eliza's But there are about 20% On the certain circumstances in certain populations that don't give you an ELISA Redoubt, but when you take the same sera and you do a protective neutralization assay in vitro
34:23
They're positive So that's you know, it's this type of discrepancy, which I find particularly interesting So yes partially that is a problem and the next example I show you is is You know in the last 10 years
34:41
Very rare cases of very early pertussis Has come up in very small children before three months of age that was unheard of Up to 20 years ago So nobody has looked into that. Why why does that happen? And the easiest prediction is the mother
35:01
Was not properly immunized or boosted to keep up her antibody titers against pertussis And those are the babies that now suffer. This is all doable, you know easy experiment clinical One question was down there if I yes and one last question, please be precise and short in your question
35:26
And then we make it in time Great talk. Thank you very much for your insight Do you think we will be able in the future to? Separate them then it's the supposed autoimmunity
35:41
From tumor immunology like now with all these checkpoint inhibitors You're curing well curing you're improving cancer or tumor Attack for the tisels, but then some patients are getting out immunity. Do you think we will maybe find like something that
36:01
Will be able to separate between the two of them like cure cancer, let's say but now get out immunity I Mean the short answer is no. I don't think this will happen because all therapeutic to anti-tumor type of immunotherapy is At its base its immunopathology or autoimmunity
36:24
It doesn't matter what you call it and we have done actually a model experiment We have used these islet cells with viral antigen a very strong tumor antigen and we have used Sarcoma cells with exactly the same to manage. So now we can have mice
36:43
That have in the islet cells that new antigen and they don't react and the tumor also expresses a Strong tumor the same and now you come in for example with dendritic cells that carry the same peptide and
37:01
You simply titrate in or you take vaccine your recombinants It doesn't matter what and you find not even a twofold difference in the titration So you always get tumor control plus autoimmunity and that dose response is identical
37:21
Last brief question. Okay. Thank you very much for that excellent presentation What I notice to illustrate your point what you use disease that really were successfully control with antibody and generally The trend is that the disease that we really struggle with nowadays like tuberculosis
37:43
Antibody were not really proven to be very effective What it's in your view the point that could be tackled in cut off tuberculosis, for example to better control the disease You know, let me say something that is complete completely politically incorrect
38:03
TB In many ways lepra the same is not a serious disease in terms of epidemiology it's a rare disease in terms of you know, Really serious TB so having said that
38:24
You know that TB always persists once you're infected the same is true for lepra and It is that granuloma with the living TB bacilli that actually creates a sort of a vaccine
38:41
Because it's a lesion That is outside of the lymphatic tissue because if it were in the lymphatic tissue You would have a more serious type of TB, but this is not really the case So now you have in a way an ongoing immune response that controls the granuloma But prevents that granuloma to open up and spread all over the place. So in a way you can say
39:06
why type TB or Leprosy is actually the optimal vaccine And by reduction you say we just haven't gotten clever enough to imitate that without any
39:22
disease consequences Stefan Kaufmann will not like that statement in its bluntness, you know But I think that's the way I think about the problems CMV is the same problem, you know
39:40
Salmonellosis is the same problem malaria is the same problem all these persisting types Just mustn't kill you before the age of 22 So, thank you