Thank you guys for inviting me here. It's hard to follow Jake and Sense of humor, so the way I There's a lot that could be talked about with this subject and I really wanted to just focus on Really the specific genomics genetics in this this situation of tolerance versus non-tolerance is there's a lot you can Look at with in terms of other markers but I really wanted to focus this as we didn't have a whole lot of time and I know that Sandy's gonna be Covering a fair amount of this interpersonalized immunosuppression talk So I guess though these are my disclosures the way I look at Tolerance, I think we can pretty reasonably well defines off immunosuppression normal graph function and but non-tolerance I kind of view as a little bit of a wastebasket by the purest sort of definition is you have somebody in a withdrawal study and they have a rejection either clinically during the course of withdrawal or you wean immunosuppression and you do a biopsy typically a year later two years later and They have what Jake is describing which is either rejection or just too much inflammation And we can use this concern that this is non Non-tolerant, but I kind of took the liberty of sort of expanding that to clinical acute rejection and we see we can sort of deem that as sort of a Form of non-tolerance in general practice. And so I wanted to kind of talk about some of the genomics signatures of acute rejection, but then also according to lots of research from Many in this room and what Jake just presented which is this subclinical graft inflammation particularly even in Enrollment biopsies for patients being considered for tolerance Inducing studies that show some inflammation and you can this is sort of a subclinical type rejection Maybe has to do with antibodies. It has to do Maybe with some other other factors. It's sort of an under immunosuppressed state and Of course, the question is what to do about those patients, but certainly there's been really interesting research on Looking at those graphs that you would think look would be okay by count biochemically, but actually have Damage inflammation and fibrosis and so I sort of think of non-tolerant In a way is when we're thinking about signatures is there can be a few different waste baskets to include that terminology so just this is just a looking at kind of genetics genomics proteomics metabolomics, so I think there's a lot of interest in sort of this middle category, which is genomics and proteomics and and And Tolerance and non-tolerance, but certainly this is a pathway you have a genetic potentially genetic predisposition then you have a transplant and Certainly the genes change according to immune activation or quiescence proteins get expressed from PBMC perhaps Maybe in the graft and then there's metabolites that can come out of that that May correlate with some of these outcomes Of course ultimately it's the phenotype that is the most important so you have tolerance you have non-tolerance Which again can be a few different categories? So just going to focusing first on real tolerance there's been several lines of evidence that there are distinct profiles of gene expression and patients who either have spontaneous tolerance meaning they are found at the off immunosuppression and they're tested or they are put into a tolerance study and at the baseline have some signs of gene expression that predict the successful or unsuccessful outcome, I think what it what is interesting is if you can see on the on the right is that there are Certainly this can be a different very different from the kidney transplant situation kidney transplanted there seems to be more of a tolerogenic state associated with B cells and So when you look at some overlaps there there seem to be little overlap between liver and kidney. It's just a very different type of immunoregulation and so this was another study from the Barcelona and UK group Alberto Sanchez Fuego who had looked at markers of immunological senescence and and If you look at some of these gene signals, they support and patients who have Who are tolerant may have some of these markers and in fact, this was an interesting study Maybe not as clinically relevant now because of these were hepatitis C infected tolerant recipients But certainly there's something about viral disease and stimulating Immunological quiescence that that can can correlate with some of these markers in the graft and This was a correlative Biomarker example of the gene expression both within PBMC and the allograft in Alberto's weaning trial that was was published near simultaneous to this paper In fact, I think this paper came out before the actual publication of the of the of the study and this is the one where it showed that patients who are older and further out from the transplant have a higher increasingly higher success rate with withdrawal, but what was looked at was in the PBMC the gene signatures and And then in the allograft and it seemed actually that the allograft itself. There was a gene signature uniquely of iron homeostasis that was that was fairly predictive of successful withdrawal of immunosuppression and so this has led to a clinical trial that has actually uniquely used this biomarker, which is the As a to see if the biomarker based approach is More you're you're able to get patients who are likely to have success and overall Will you be able to see a higher success rate by using this liver tissue based biomarker? And so it'll be interesting to see the results. I know there's been some difficulty with this trial, but Certainly this type of design is really how you can make use a biomarker like this and prove that using it to Select patients for withdrawal is the way to go compared to just a weaning all approach Where we know that there are certain predictors of that and we certainly want while it may be safe We certainly want to be able to select patients better for these for these strategies So one of the I wanted to kind of talk a little bit about some of the gene signatures that we've been looking at with mTOR inhibitors and it turns out that if you convert patients from a CNI to serolimus Tacrolimus to serolimus there is a profile that direct conversion Within the blood and we also saw this in the bone marrow, but in the gene at the gene expression In the peripheral blood you see actually a conversion into more of a tolerogenic gene expression pathway, so some of these pathways that appear to change with just that conversion are regulatory t-cell Pathways Fox p3 gene expression things like that when you and this goes along with mTOR inhibitors perhaps being pro-regulatory and so we took that approach and Weaned patients off of a small cohort at our center Weaned a cohort and we had about a little bit over 50% success rate of weaning off of serolimus and we were able to actually find some blood probe sets that distinguished patients at baseline from That would go on to become tolerant or non-tolerant and we just recently found out last week this was accepted in hepatology our paper and Very I think one of the sort of coolest things that we found we looked at the biopsy signatures We actually had some differences between tolerant and non-tolerant, but we sent these Tissue to Alberto to look at his Gene signature that he's using in his clinical trial that grew before and it actually Predicted tolerance in 85% of these patients So it seems to be maybe the signature is independent of the immunosuppression used That it actually may work in CNI and also mTOR inhibitor treated patients so just moving to towards kind of non-tolerance where I sort of look at this as a kind of rejection immune activation biomarkers And if you look at the profile many of these are related to genetic polymorphisms however, I think more recently for Potentially clinical practice use there are a micro RNA and mRNA and proteo form panels that may be able to be utilized to monitor paper standard patients who are undergoing immunosuppression modifications and This is obvious data looking at these were Patients actually in the in a tolerance study in a wish but these were these were before they Were at the marker where they were going to be weaned off immunosuppression within the first year and you can see that there's a pretty high prediction of a micro RNA signature that can predict Rejection and even in in 50 to 100 days prior this signature seems to increase prior to that compared to patients with non rejection We have done a couple of areas we have a similar area of interest and we're looking at this in a larger grant of Protea forms using top-down proteomics and we found specific protea forms and these are specific to PBM C's From patients with rejection versus healthy transplant versus dysfunction without rejection We're looking to validate a few of these protea forms that could be even perhaps more specific than general blood mRNA expression and I just presented this yesterday This is our C Todd Northwestern and C Todd 14 Study and it very looks very similar to obvious data where you can see a rise in a gene expression signature in the peripheral blood 36 probes that increase slowly before rejection, but I think more potentially Clinically relevant is that there is in a stable transplant course post transplant you see healthy transplant The gene expression scores go down over time as they're serially being followed and that may be helpful to use as a minimization test This is what we're developing and so I'm going to end a little bit with the the I think Jake touched on this which is T CMR T-cell mediated rejection transcriptional signatures that You actually can see during immunosuppression withdrawal or even at baseline And so this classifier that was developed seems to perhaps be Is associated with T cell mediated rejection and in in Sandy's I would study which I'm sure she will talk about it in more detail And Jake mentioned this that there were there were of significant interest Is this category of pediatric recipients that were non-eligible for immunosuppression withdrawal? and if you look at there's a breakdown again of having inflammation fibrosis or neither and and When you look at these transcriptional pathways, they actually These T cell mediated rejection pathways seemed enriched in those who have interface activity and very similar to T-cell mediated rejection and these are the different of the clusters and so the molecular profile of a subset of biopsies with interface Activity overlaps with T cell mediated rejection So when you look at these patients that you perhaps do a protocol biopsy on I know that this is fair often done in pediatric liver transplant practice, but adult Liver transplant programs are catching on in fact There there's sort of been a I can see a resurgence in protocol biopsies perhaps in younger Adults who are transitioned and we certainly seeing more interest in fatty liver recurrent NASH and sometimes you see things like this and it seems like maybe this inflammation is really really kind of a type of Rejection that needs to be treated more aggressively. And so that's similar to the adult work too is where you see this this gene expression and maybe there's some overlap with With markers of fibrosis and you can see the high expression level seen in patients who have higher APRI scores lower platelet counts, so In just in conclusion if we look at genomic signatures of tolerance, I think this liver biopsy profile is very interesting it seems to maybe be predictive of pre-withdrawal of Independent of immunosuppression therapy. It seems relatively robust, but it certainly needs to be proven And then genomic signatures of non-tolerance We're very interested. I think in this actual clinic T's a clinical T cell media rejection to be able to monitor patients But particularly to pick up some of these signs before there's graft injury to be able to modify immunosuppression to avoid these this the occurrence of rejection particularly with the interest in more minimization strategies and it seems like this subclinical inflammation may be related to T cell media rejection based on the molecular profile. So ideally it would be using These strategies in clinical practice. We're not there yet, but we're getting I think closer and closer to have biopsy based peripheral blood markers That can that could perhaps be used to guide our current management Thank you

genomics

genetics

tolerance

transplant recipients

immunosuppression withdrawal