0:00
Thank you for the kind introductions.
0:02
So actually I'm a research group lead at the Technical University of Munich and I'm heavily working with the Atlas system that is provided by Nanion.
0:13
So what I will present today is basically my results from our research and how we use this technology in order to address our questions.
0:24
So I choose the title to say that we start to assess tumour susceptibility against antigen specific and antigen unspecific CD8 T cells.
0:37
And when I did my PhD in at the same institute, I started to understand why CD8 T cells are detrimental to tissue.
0:47
Now I shift my attention how we can use the pathogenic potential of these T cells to be beneficial in the tumour context.
0:55
So that's why I will also start with a background how the tumour and micro environment can influence effective function of CD8 T cells could be various factors.
1:06
I mean all of you are very aware of that.
1:09
So it's the nutrient availability, it's the expression of Co inhibitor receptors on T cells that's can be the cytokines in the micro environment.
1:17
But what I would like to focus today in this topic and this presentation is more about the antigen because the discovery that we made some years ago deals with an antigen independent mechanisms of killing of target cells.
1:34
Because tumour cells, as we know, have low levels of MHC Class 1 molecules that can present antigens or the identity of antigens presented by tumour cells are not as immunogenic, people started to think about alternatives.
1:47
They generate CARs, TCRs, artificially introduced to T cells, and then they can recognise antigens that are not presented on MHC and are really effective in eliminating tumour cells.
2:03
But I would like to introduce another layer of CD8 T cytotoxicity which is independent of an antigen recognition.
2:12
And this is based on this discovery which we made four years ago in the context of fatty liver disease, where we could see that there are CD8 T cells in the steroid inflammatory context that can sense metabolites in the microenvironment that activate these T cells independent of an MHC Class 1 mechanisms.
2:32
And we call this killing CD8 T cell or depression because at that time it was kind of an autoimmune like a phenotype.
2:41
But still these CD8 T cells can do their job with the TCR, but on top, they acquire a function that is a state that allows them to kill target cells, MHC class independent.
2:53
And this is a process licenced by IL-15.
2:56
So we shifted our intention from fatty liver disease to other human very relevant disease and inflammatory bowel disease.
3:05
And we wanted to know whether although also in these patients we do find T cells that kill target cells in MHC class one independent fashion.
3:16
So our overall setup in our group is to use K562 cells, which are MHC Class 1 deficient cells.
3:23
And what we did here is we isolated CD8 T cells from an inflamed tissue of IBD patients and compared those T cells that were isolated from healthy tissue or from blood.
3:33
And what you can see that only in the tissue of inflamed of IBD patient with an inflamed tissue, you find CD8 T cells that can kill target cells in MHC class one independent fashion.
3:45
So this wasn't facts based killing because the numbers that we got from the tissue was very low and we wanted to understand this killing dynamics and capacity in more detail and that's why we shifted our focus more in vitro setup.
3:59
And what we do know is that IL-15 is an important cytokine driving this process.
4:05
So to really understand the dynamics of the killing in vitro, I use very a lot like an impedance based technology because this is a technology where we can measure in real time the capacity, the killing of target cells.
4:26
And it's important that we have an electrical current that is delivered to the electrodes that can pass this electrode cell interface, which you can see here in this graph.
4:37
So you can by measuring the impedance and know whether a cell increase its cell attention, its proliferation or what is important for our purpose is whether it detached from the bottom as a proxy for cell killing, target cell killing.
4:55
And this is what we usually do our assays.
4:58
So we seed our tumour cells, whether they're adherent or non-adherent.
5:03
The non adherent cells we coated plate with an antibody to make them adherent and then measure the killing of target cells.
5:13
So first experiment that we did with this device is here you see the workflow what we usually do isolate the apheresis from healthy donor, we treat them with IL-15 for two days and to know whether these cells have a permanent ability of killing in an MHC class one independent fashion or this is only transient.
5:34
So we check this by removing the IL-15 stimulus again and leave them without the stimulus for two days and checked whether they also lose their killing capacity.
5:45
We do see this here in this graph that if you have permanent IL-15 stimulation, the cells really nicely kill the K562 cells. Once remove the IL-15 again, they also lose the capacity to kill in an MHC class one independent fashion.
6:03
So this is as we call it as a transient cytotoxic seeded T cells stated these T cells upon IL-15 exposure can acquire and lose and the IL-15 signal is lost.
6:16
And interestingly, when we compare their killing capacity to like really the prototypic innate cytotoxic lymphocytes, the NK cells and were also treated with IL-15 and we somehow normalised the number of really the ones that can kill target cells.
6:33
Then they have kind of a similar killing dynamics, which was really intriguing to us. Now because OK, where how these T cells then gets activated?
6:47
So people thought and this is still a valid NKG2D is also a very important surface receptor that can mediate MHC class one independent killing.
6:58
But we have not seen such strong effects when we were blocking this ligand, this receptor and then we were searching for other potential stimulator can activate T cells.
7:13
In our Nature paper, we have identified metabolites that were ATP and NAD, which you can appreciate was not the case for humans that T cells.
7:20
But what we can see instead was that actually the histones that are released from the target cells are able to induce degranulation of IL-15 licenced CD8 T cell, which was really striking because we would not have expect to have histones as a potential T cell activation stimuli, A stimulus that can elicit degranulation of CD8 T cells.
7:46
So what we did also to get kind of a sense, OK, histones, so they usually are considered as stamps that can activate innate immune cells, but not CD8 T cells.
8:01
We were checking for the abundance of histones in the lysates so intercellularly and also in the supernatant to see whether they prominently exist.
8:13
They prominently exist in the supernatant.
8:15
So here you can see that within the cell they're highly abundant.
8:19
So it's like the like one of the top hits in the lysate you find histones, but you also see them in the supernatant of normal non dying proliferating K562 cells.
8:29
So somehow and the reason for it we don't know yet, they also secrete the histones.
8:34
I mean these are transformed cells that are not like normal healthy primary cells in the normal in my environment in the body.
8:41
So I don't know where whether also normal cells can secrete it.
8:45
But in our purposes, the histones are really abundant in the supernatant, and I think by that they can also nicely activate T cells if they are in close contact.
8:56
So I think this could be also another alternative thinking of how tumour cells can also get killed independent of tumour antigen presented by them.
9:07
So that's what I would like to further elucidate on whether this pathway might be important for and cancer therapy.
9:18
So this is a small scheme.
9:20
So we know IL-15 is in the beginning for the licence important.
9:25
Then we have when they come together the target cell and the CD8 T cell histones that can trigger receptors and a pathway which are not able to disclose because this is now a patent that is on that we have file right now.
9:37
But what I can provide is that when we use an inhibitor against this pathway and see whether there is a pathway that is specific for this cytotoxic function, then we can also use this device to check for.
9:56
And as you can see, when we use an inhibitor that is specifically inhibiting this pathway, this histone triggered signalling pathway, which I called, then you can nicely block this killing of CD8 T cells.
10:09
So we're lysing with IL-15 compared to NK cells.
10:11
So this is a real clear discrimination and interestingly enough, so when we use an antigen specific model, so in this case these are CARs either directed against in a fibroblast target cell line or EGFR against a pancreatic cell line.
10:30
Then when we use the same inhibitor with the same concentration, we were not able to block it.
10:35
So clearly there's a discrimination of a pathway in these cells that is operative for recognising histones and eliciting an MHC class independent killing.
10:48
But it's redundant if you have an antigen specific.
10:52
So it's not interfering with the CAR T cell killing, which is really promising because I mean you don't want to have an emitter that blocks CAR T cell killing or antigen specific killing.
11:03
So leaves the pathogen potential pathogenic cytotoxicity intact, interfere with it.
11:12
So as I said in the beginning, so this was data where we wanted to know whether the seeded T cells that can potentially cause tissue damage.
11:22
Now in current projects what we are running is whether we can make use of it for efficient anti-cancer immunity.
11:29
So to give you an example, whether we do find CD8 T cells in the tumour microenvironment that when you put them out of the hostile microenvironment use put them into a in vitro setting where you just don't have any this hostile factors.
11:44
Indeed, you will find CD8 T cells in the tumour tissue that are able to kill target cells in an MHC class one independent fashion.
11:51
So potentially there are cells that are very effective.
11:57
So what we did we so in the in this paper and in our manuscript, we defined a list of genes that can it’s the best proxy for us on a transcription level to identify cells with this potential function.
12:13
So with this list, we went through the single celled RNC data from several studies where they analysed cohorts of patients that were treated with ICB.
12:23
In this case, I don't know in general it was ICB, anti CD4 and this was anti PD1 to see whether we do find CD8 T cells in these cohorts with this gene signature.
12:36
So what you handed, she isolated the T cells from the, so extracted the T cells from the single cell data.
12:42
So this is UCell score for this list of genes that we used to define these cells in this subset and this UCell in the single cell data set.
12:52
And you can see that there are cells that have a high score for this gene signature.
12:59
And interestingly enough, the frequency of cells that have a high score for this signature and nicely correlate with the ICB efficacy.
13:10
So if you have more of these cells before the treatment, then the likelihood to have complete response.
13:16
So CR compared to partial and steady disease is higher.
13:19
I mean it's still 22 patients, but still, there's already a good hint that also antigen independent potential antigen independent mechanisms are important.
13:31
I mean this we can't prove not yet.
13:34
But I think there's also beyond antigen specific killing also some important contribution of antigen unspecific killing.
13:44
We also were interested in whether there's any hint for in tumour, whether some tumours might react or be more susceptible for this kind of tumour cell killing.
13:57
So what you handed is also to check the tumour cells in these data sets, whether there are any gene sets, enrichment pathways that are enriched for better ICB efficacy.
14:07
So he combined all three data sets together and what came out at and mostly he finds a signature for an interferon alpha response.
14:15
So if you have a higher interferon alpha response, they'll more likely respond better to ICB, which makes sense because interferon alpha, it decreases MHC Class 1 molecules.
14:26
You have more antigens presented and it's better to kill target cells.
14:31
So to check whether this is really the case in our setting, so we use A549 wild type mice and also not mice, sorry, cell lines.
14:40
And when we also knocked out the B2M, which get rid of the HLA type 1 molecules on these cells, we pretreat them with interferon alpha for 24 hours.
14:52
The first thing, what was really striking to us, it doesn't matter whether you have the HLA or not.
14:57
So an mismatch reaction or an HLA that is not present.
15:02
So I have the data here still you have similar efficacy of killing whether it's HLA deficient or not, which is also very interesting.
15:12
But then if you apply interferon alpha, you get a massive increase of HLA type one on these cells, which is expected and you don't get it within this knockout cell.
15:22
And so these cells are really devoid of any HLA type 1 molecules.
15:26
And then if you then add the IL-15 simulated CD8 T cells and said you get even increased killing of it which was not as prominent as you have the HLA type competent A549 cells.
15:38
So that means that it I think that there is some contribution also of antigen specific killing in the tumour context, which I would further like to elucidate.
15:52
And this difference we could also capture by the killing by the Atlas systems where we check the dynamics not like endpoints, but also over the time.
16:04
So you can clearly see that there's also this difference between whether you have an HLA mismatch killing or you have no HLA mismatch killing.
16:14
So we call it an auto aggressive killing, which is dependent on the histone triggered signalling pathway.
16:22
And with this, I'm at the end.
16:25
So this is the summary.
16:26
So I hope you could appreciate that this platform, which this impedance based platform is a valid and nice tool to really capture the killing dynamics of antigen dependent and antigen independent killing modes of the CD8 T cells.
16:45
I could, I showed you a potential novel antigen dependent mechanisms of killing target cells that we've start now to also further elucidate in the tumour context.
16:59
And I think that it might be also another important layer of, yeah, optimising anti-cancer immunity, whether we can think of exploiting this antigen independent killing in a more efficient way.
17:15
Also in for further therapies.
17:19
And yeah, this is my group.
17:21
So I'm at the institute of molecular immunology my mentor’s Percy Knolle.
17:25
I have also my own group.
17:26
We have nice collaborations within the institute.
17:30
I want to thank then the group of Nanion which was really big help in setting up the machine and also giving advice as how to nicely use the machine.
17:42
And I want to thank you for attention and if you have any further questions, you can visit us at booth #13 and we are staying here for the next hours and happy to chat with you.
17:52
Thank you very much.