Podcasts about hla a2

This week, we're hearing about lung cancer data from the ESMO Congress 2021—specifically, a novel tyrosine kinase inhibitor for HER2 exon 20–mutated non–small cell lung cancer, and a second-line vaccine for HLA-A2–positive disease.To listen to more podcasts from ASCO, visit asco.org/podcasts.

Effective control of acute myeloid leukaemia and acute lymphoblastic leukaemia progression by telomerase specific adoptive T-cell therapy Full Text- https://tinyurl.com/yb3gjwqs Telomerase (TERT) is a ribonucleoprotein enzyme that preserves the molecular organization at the ends of eukaryotic chromosomes. Since TERT deregulation is a common step in leukaemia, treatments targeting telomerase might be useful for the therapy of hematologic malignancies. Despite a large spectrum of potential drugs, their bench-to-bedside translation is quite limited, with only a therapeutic vaccine in the clinic and a telomerase inhibitor at late stage of preclinical validation. We recently demonstrated that the adoptive transfer of T cell transduced with an HLA-A2-restricted T-cell receptor (TCR), which recognize human TERT with high avidity, controls human B-cell chronic lymphocytic leukaemia (B-CLL) progression without severe side-effects in humanized mice. In the present report, we show the ability of our approach to limit the progression of more aggressive leukemic pathologies, such as acute myeloid leukaemia (AML) and B-cell acute lymphoblastic leukaemia (B-ALL). Together, our findings demonstrate that TERT-based adoptive cell therapy is a concrete platform of T cell-mediated immunotherapy for leukaemia treatment. Facebook - bit.ly/2xznxjV Twitter - bit.ly/2xzWvsu LinkedIn - bit.ly/2xzJ6kc Pintrest - bit.ly/2xzX8SS Reddit - bit.ly/2hoxI0N www.Oncotarget.com

[intro music]   Host – Dan Keller Hello, and welcome to Episode Fourteen of Multiple Sclerosis Discovery, the podcast of the MS Discovery Forum. I’m your host, Dan Keller.   This week’s podcast features part two of an interview with Professor Gavin Giovannoni about the role of Epstein-Barr virus in MS. But to begin, here is a brief summary of some of the latest developments on the MS Discovery Forum at msdiscovery.org.   We've published a blog post from Christine Granfield, the founder of HealthCare Journey. Healthcarejourney.org is a website designed to help MS patients easily navigate the expanse of information about MS. In the blog post, Ms. Granfield says that HealthCare Journey is not meant to replace the doctor-patient relationship but instead provide a place where patients can find answers to questions with accurate, up-to-date information when their physician might not be available.   We published another blog post written by our intern, Cynthia McKelvey, on how to interact with the news media. News about science is often sensationalized and over simplified. But that’s all the more reason for researchers and clinicians to be media-friendly. In her post, Cynthia offers 13 tips on how to talk to reporters so you can be sure the best and most accurate information gets to the public.   Last week we interviewed Dr. Alan Thompson of the International Progressive MS Alliance about the difficulties of researching progressive MS. We published more information on the Alliance, its goals, and a full list of the 22 recipients for their first round of funding, totaling 22 million euros. To view the article, visit msdiscovery.org and visit our News Briefs section under the News and Future Directions tab.   [transition music]   Now to the interview. Professor Gavin Giovannoni from Queen Mary University in London is one of the worlds most prolific and most visible MS researchers and clinicians. He's also on the scientific advisory board for MSDF. Two weeks ago we featured the first part of my interview with him about the potential for a cure in MS. This week we’re going to discuss the roll of EBV in MS.   Interviewer – Dan Keller Professor Giovannoni, where does it come in? How has it entered the thinking?   Interviewee – Gavin Giovannoni We have a causal theory. We don't just switch on a light switch, and we've got a call that said it evolves over time. And if there's one particular thing that looks like it's got the top candidate for being a cause of MS is Epstein-Barr virus. And I think the original observation is epidemiological. People with infectious mononucleosis, which is delayed EBV infection that is symptomatic, have a higher risk of developing MS, and that's been reproduced now across many, many studies as a risk factor. But EBV infection in itself is a risk factor because if you're not infected with the virus, in other words people who don't have the virus, have a very, very low risk…it's almost a zero risk of getting the disease. So in terms of its negative predictive value – that's the strongest value we have – people don't get MS if they don't have EBV. So it looks like it’s an essential component of the causal pathway. How it's acting in the causal pathway? We don't have any idea. We just do know that if you do get EBV infection or infectious mono it's a risk factor throughout life. So in the Danish study, it shows the risk remains even when you go into your 40s, 50s, and 60s, which is interesting.   And EBV is a complex biology. We don't know where it's acting. We know it resides as a latent infection in the B cell. And what it's doing to B-cell biology is incredibly complicated. It hijacks B-cell biology, and it affects its antigen-presenting function, it affects its survival, etc. So people are targeting the B cell; I think the B cell is important. And the reason why I think the B cell is important when you look at all the most effective therapies in MS, when you put them on a chart with all the cells they affect, the only common cell to all of them is the B cell. And the link that the B cell may be the Epstein-Barr virus. And how EBV triggers autoimmunity haven't a clue. We originally thought well maybe the link between infectious mono and MS was related to HLA susceptibility. But we did a study on university students in the UK, and we showed that the HLA type that predisposes you to infectious mono is not the HLA type that predisposes you to MS. So we don't think it's at the HLA level that EBV is interacting with MS susceptibility; it's somewhere else.   The other risk factors are smoking. And when you start putting smoking and EBV and low vitamin D, which is the other environmental factor, together it looks like there's some interaction of all three components. But how they're working at a biological level that's where research needs to be done. So some people are … I think there's mimicry between EBV and myelin proteins, and there is some data that there are some antigenic epitopes in the EBV proteins and [?] antigens, which is why a lot of people are focused on CD4 cells as being the link. I'm not sure if it is the CD4. Others are focused on the CD8 cells as being the link. Michael Pender in Brisbane, Australia, thinks that Epstein-Barr virus is acting as the innate stimulus, the danger signal, that just upregulates innate immunity that then allows autoimmune responses to occur on top of that. And he thinks that's occurring in the central nervous system. That's his theory. And he thinks that people with multiple sclerosis have a deficiency of cytotoxic CD8 cells that keep the EBV virus in check. So he's now testing the strategy of trying to boost the CD8 response against EBV and kill the Epstein-Barr virus to lower the innate activation and reduce autoimmunity. That's his theory.   I wish I could tell him my theory. All I know is that the epidemiological observations are pretty standing. And you know, we probably should be doing a vaccine trial to test the hypothesis. There is a vaccine for EBV, but it's been discontinued. So GSK developed the vaccine. It wasn't that effective in stopping EBV infection, but it was very effective in preventing infectious mono. So what it did was it raised your immunity to a level that stopped you getting infectious mono, and that may be enough for MS prevention. You know, when they sold the EBV vaccine program to MedImmune, and I heard about six months ago that maybe they had stopped the program. There is no EBV vaccine program occurring in the pharmaceutical arena. There's been a recent meeting in Oxford around Epstein's anniversary because it's 50 years since he discovered the virus. And the, Harold Varmus was there, and there's a big push now for the NIH to fund a vaccine study – interesting not to prevent MS but to prevent the secondary malignancies linked to EBV. Because EBV is linked to a whole lot of hematological malignancies. So the idea there would be if you could prevent people getting Epstein-Barr you prevent a whole lot of lymphomas particularly. I'm personally a little worried about that strategy because EBV is one of our most co-evolved viruses. At a population level, it's part of our immune systems. So I actually think at a population level EBV must be doing something good for society and the population. I think it may be a link to B-cell memory or something like that.   So if we stop people getting EBV, we may be storing our problems at a population level. But until we do the trials we won't know. So we need a vaccine, and that's the way to test is EBV causal? Coming away from it, it may just be the trigger, or it may be driving the disease continuous. If that's the case, then we need to have anti-EBV drugs. And there is one being tried right now; it's called ocrelizumab. It's an anti-CD20 drug. Itself hasn't been tested as an anti-EBV drug, its predecessor, which is rituximab, is licensed as an anti-EBV. It's actually the only drug that's licensed to treat EBV. It's licensed to treat EBV associated lymphoproliferative disease, which occurs in transplant patients. It's pretty effective at switching off on that condition, and the EBV levels just plummet. A company that's developing ocrelizumab, which is Genentech Roche, wouldn’t like for me to say that it's an anti-EBV drug, but that's exactly how it may be working in MS; it may be targeting EBV. The obvious thing is to test antivirals that target EBV. There are no specific ones that have been designed for EBV, but we've got a particular drug that we would love to test against EBV because it has some activity. Trying to get the funders convinced that we should do a trial of an anti-EBV drug in MS has been difficult. I think we shouldn't ignore the EBV hypothesis, though, because the data out there is pretty compelling that it's causal. And as a community, I think we have a responsibility to test whether or not it is causal. And the only way we can do that is intervention studies – vaccines and targeting the virus with antibiotics.   MSDF It seems the geographic distribution of MS may actually be opposite say the distribution of Burkitt’s lymphoma. And what is EBV doing and how does it do it in different regions? And I wonder if that brings in the vitamin D hypothesis again.   Dr. Giovannoni MS prevalence pretty much matches infectious mono prevalence. So infectious mono has also got a gradient. The Burkitt's lymphoma thing probably that follows patterns of parasitic infection, particularly in malaria. So I think EBV probably interacts with other infectious agents, and that's one of the theories about EBV; it's not working on its own; it's working as a coinfection with another virus. The other virus that we need to talk about are the family of the HERVs, human endogenous retroviruses. Because the EBV is a potent transactivator of these viruses. In the big body of literature on HERVs being involved in MS, a lot of us think it's associative. In other words, inflammatory response triggers transactivation of HERVs in what we see as an epiphenomenon. But there are people who think it may be linked to the cause of the disease. Again the only way we can test this hypothesis is by treating people with drugs that target EBV and HERVs. Because HERVs are drugable; they are retroviruses, and you've got a whole arsenal of therapy that could target the various components of HERV biology. We should be doing trials in that as well.   Coming back to the vitamin D, there has been one small study that needs to be reproduced showing that if you do get EBV infection when your vitamin D levels are low your antibody responses are much more marked. And so there may be some link between low vitamin D and infection, but nobody has actually studied that formally, and I think it's something we tried to do with our epidemiological tools to see vitamin D deficiency or low levels makes it more likely that you're going to get infectious mono, that maybe they are interacting with each other. I don't think it's going to be as simple as that, though, to be honest with you. I think they're probably going to be working in an immunological level. I'm not sure if they're going to be causal; I think they may be associated with each other. EBV triggers a mess of lymphoid proliferation, which consumes vitamin D levels. So if you find someone with infectious mono and they have low D levels, it could be the infectious mono is reverse causation rather than the other way of causation. So we need to do that prospectively, and it's a difficult study to do.   But I think also the other thing you've got to look at is when they're starting to put all of these risk factors together in studies, and this has been mainly been done in the Scandinavian databases – and you start putting the HLA-DR15 in, the protective HLA-A2 in, the history of infectious mono, serum levels of anti-EBV antibodies, put smoking in, start putting vitamin D levels in – you're beginning to see relative up about 40. So that's a big signal to me because the doyen of causation theory, Bradford Hill, said that when you start getting relative risks above 40 that you should be thinking causation. So I'm beginning to see a causal pathway where all of these factors now are giving relative risks that are very high compared to the background population. And so, those factors must all be in the causal pathway. And the question is which one can you intervene in? And there's a few you can intervene in. But EBV is the obvious one. If you take it out of the causal pathway, you may be able to prevent this disease.   So I'm lobbying – and whole lot of us are lobbying – that we really need to get the public health community and the MS community and the virology community together so we can start thinking about prevention trials. Around about 5 to 10% of the adult population are EBV negative. But that group of people don't get MS. So that's the important factor is those people don’t get MS.   MSDF But the flip side is 90% of the population has been exposed to EBV, and most of those people don't get MS. So do you think it's all of these other cofactors you've mentioned? Whereas EBV sort of lights the fire if the fuel is there?   Dr. Giovannoni Yes, I think that's true. And Bradford Hill, I mean he developed his theory for causation around common manifestations or re-exposures. Like asbestosis is one of the examples he always used that if you had asbestosis, which is quite of a rare exposure, the chances of getting mesothelioma, which is a rare cancer, was almost 200 times background. So that's easy to understand. But EBV is such a common exposure, and MS is a relatively rare manifestation. So in that situation, the relative risks come down. So even though the relative risk of getting MS is only about 2.2 to 2.5 with infectious mono, it doesn't mean to say it's not causal. It could still be causal. So I'm not worried that the relative risks are low. But I agree with if it's EBV infection on top of something else the genetic susceptibility, the low vitamin D, or all those other factors that then triggers the autoimmune response.   But if EBV is a pivotal factor and you stop it, those other factors are irrelevant. That's why we need to do prevention studies. We need the vaccine, though. The original vaccine strategy was developed to prevent EBV completely; that's in terms of oncoprevention. But in terms of MS and autoimmunity, you may not have to prevent Epstein-Barr virus. Maybe what you need is to make sure you're vitamin D replete and get wild-type infection when you're very young. That may be sufficient to lower the risk of MS. You may not necessarily need a lifelong protective immunity against Epstein-Barr, but maybe you just need to be infected at a young age when your immune system can deal with it. It doesn't fertilize the field for autoimmunity later on in life; that may be the strategy.   MSDF Does it get into the possibility of tolerance?   Dr. Giovannoni Yes.   MSDF To EBV?   Dr. Giovannoni If we knew about the biology of EBV, I mean I'm not a virologist. And when I delve into the complex biology of Epstein-Barr virus, how it's fooling the B cell into surviving longer and bypasses B-cell signaling pathways, it's a credibly cleaver virus. And there's a lot of biology there that needs to be picked apart in the MS field. So there may be pathways in the EBV biological pathway that could be targeted rather than just targeting the virus itself. So I think there's lots of research to be done. This is why we, as an MS community, really need to bring virologists into the community as much as possible. And at the moment, that doesn't seem to be happening a lot. There's very few virologists interested in MS. You can count them on one hand to be honest with you that are really interested in MS. Most virologists are working on other diseases. And when you go to them, it's hard to get them interested in MS because MS is something on the periphery of their thought. They're usually targeting obviously infectious diseases. But most of the EBV virologists are working in oncology, lymphomas.   MSDF Very good. Thank you.   [transition music]   MSDF Thank you for listening to Episode Fourteen of Multiple Sclerosis Discovery. This podcast was produced by the MS Discovery Forum, MSDF, the premier source of independent news and information on MS research. MSDF’s executive editor is Robert Finn. Msdiscovery.org is part of the non-profit Accelerated Cure Project for Multiple Sclerosis. Robert McBurney is our President and CEO, and Hollie Schmidt is vice president of scientific operations.   Msdiscovery.org aims to focus attention on what is known and not yet known about the causes of MS and related conditions, their pathological mechanisms, and potential ways to intervene. By communicating this information in a way that builds bridges among different disciplines, we hope to open new routes toward significant clinical advances.   We’re interested in your opinions. Please join the discussion on one of our online forums or send comments, criticisms, and suggestions to editor@msdiscovery.org.   [outro music]          

Multiple Sclerosis is most probably an autoimmune inflammatory disease of the central nervous system. Demyelination of neurons and axonal loss occur in temporal and spatial resolution in multiple areas of the brain and spinal cord. This impairment manifests in neurological symptoms. The course of disease varies between individuals and the causing mechanisms still remain elusive. Environmental factors as well as genetic predispositions are widely discussed. Recent studies stressed the prominent role of CD8+ T lymphocytes in the autoimmune pathomechanism. They notably exceed the amount of CD4+ T cells in acute lesions yet target cells and activating antigens remain elusive. A genetic linkage between the human leukocyte antigen gene locus and disease susceptibility was observed. Carrying the HLA A*0301 allele or the HLA A*0201 allele correlates with a risk factor or protective effect for Multiple Sclerosis susceptibility, respectively. Thus during this thesis two main questions were followed: 1. Which CD8+ T lymphocytes participate in the autoimmune attack on central nervous system tissue and what are their receptors for antigen recognition? 2. How does the expression of HLA-A2 molecules lead to a decreased disease susceptibility? In the first part, the T cell receptor molecules of potentially disease-related single CD8+ T cells from frozen patient tissue samples were characterized. T lymphocytes were considered disease-related when they either belonged to a clonally expanded T cell population or expressed an activation marker on their cell surface. In a clone-specific approach, the T cell receptor beta chains of pre-analyzed, clonally expanded T cell populations were investigated. Further an unbiased approach independent of pre-analyses was established. In the second part, antigen recognition of the probably disease-related T cell receptor 2D1 was investigated. This receptor was isolated from a Multiple Sclerosis patient and was known to be activated by a myelin-derived peptide presented on HLA-A3 molecules. In an animal model double-transgenic mice expressing HLA-A3 and the 2D1 T cell receptor developed a Multiple Sclerosis-like disease after immunization with the known peptide. Surprisingly not a single triple-transgenic mouse expressing HLA-A3, the T cell receptor 2D1 and HLA-A2 showed symptoms after immunization. In these mice 2D1 T lymphocytes were shown to be depleted in the thymus. To characterize HLA-A2-bound peptides which mediate this protective effect a novel technology for unbiased identification of antigenic peptides recognized by human leukocyte antigen class I-restricted T lymphocytes was employed. 28 peptides presented on HLA-A2 molecules were found to be recognized by the T cell receptor 2D1. Those peptides displayed very closely related sequences. Eight possible parent proteins existing in mouse, therefrom even four equally expressed in humans were identified. Finally those putative parent proteins were further characterized and first investigations of antigen processing in different antigen presenting cell lines were performed.

Immunotherapy using T cells is a new approach that is being explored for the treatment of metastatic melanoma. For renal cell carcinoma (RCC), adoptive T cell therapy (ATT) is currently hampered by the lack of T cells expressing suitable T cell receptors (TCR). A tumor-infiltrating T cell population (TIL) was identified in our group (TIL-53) that showed a pattern of tumor recognition consistent with the requirements of a TCR applicable for immunotherapy. With the advent of genetic TCR engineering it was possible to further define the TIL specificity which was previously precluded by the failure to cultivate TIL-53. To achieve high expression levels and functionality, the TCR53 required the exchange of the human TCR constant region by the TCR murine constant region (TCR53m). The B3Z T cell hybridoma which stably expressed TCR53m after retroviral transduction was used to analyze a large panel of tumor lines and non-malignant cell cultures for expression of the TCR53 ligand. The analysis included 34 RCC cell lines, 55 tumor cell lines of different histologies and 30 non-tumor cell lines. 65 % of the HLA-A2+ RCC cells and 25 % of other HLA-A2+ tumor lines were recognized by the B3Z-TCR53m cells. Among the non-RCC tumors, the TCR53 ligand was frequently found in malignant B cell lines and EBV-transformed B-lymphoblastoid cell lines (5/13, 38 %). Of 25 HLA-A2+ non-tumor cells only 2 were marginally recognized. The TCR53 ligand expression could be increased with IFN-a but not IFN-g treatment on cell lines that already had some TCR53 ligand expression. De novo induction in cell lines that had no prior expression of the TCR53 ligand was not observed. The B3Z-TCR53m cell line could detect the TCR53 ligand on fresh tumor material and, if used for therapy, B3Z-TCR53m could be used to identify those patients whose tumors are positive for the TCR53 ligand and thus could benefit from the therapy. To achieve high expression levels and functionality of the TCR53m on human PBLs, the TCR53 a and b chain sequences had to be optimized for codon usage. PBLs expressing these recombinant TCR sequences (TCR53mc) showed very low formation of hybrid TCRs between the TCR53mc b chain and endogenous TCR a chains. TCR53mc-expressing T cells of RCC patients and healthy donors showed specific killing of tumor cell lines and had a polyfunctional profile, defined by the detection of T cells that simultaneously secreted cytokines (IFN-g, TNF-a or IL-2) and performed granule exocytosis when recognizing targets. The functional response of TCR53mc-expressing T cells depended on the expression of HLA-A2 on the target cells. Analysis of RCC tumors using multicolor fluorescence immunohistology allowed the detection and localization of CD8+ T cells in relation to blood vessels. The majority of CD8+ T cells were found extra-luminal, indicating strong extravasation of T cells into RCC tumors. The majority of the T cells in the lumen of the blood vessel had perforin (~ 90 %), while T cells that were outside the blood vessels were to a large percentage perforin negative (~ 60 %). Thus, CD8+ T cells apparently arrive at the tumor being perforin-positive and lose perforin when outside the blood vessels. The three-dimensional growth of cells in spheroids was used to mimic the tumor milieu in vitro and to evaluate the functional capacity of T cells with transgenic RCC specific TCR expression. T cells infiltrated the spheroids and preferentially accumulated in the rim of the spheroid (~ 100 μm). The killing capacity of TCR53mcexpressing T cells in the 3-D environment in a 4 h assay was similar to that observed in a standard 4 h chromium release assay with RCC cells in suspension. However, after being cultured for 24 h in the spheroids, the T cells were no longer able to secrete cytokines upon stimulation with target cells and were negative for perforin, granzyme B and CD28. The presence of CD4+ T cells in the spheroids significantly increased the number of CD8+ T cells infiltrating the 3-D tumors. Moreover, the CD8+ T cell response was enhanced with more degranulating T cells and T cells secreting cytokines, which was not seen in the absence of CD4+ T cells. The functional improvement of the CD8+ T cell response required the CD4+ T cells to be activated, as it was not observed when CD4+ T cells were used that lacked TCRs specific for the spheroid tumor cells.

Das PHLDA1 (pleckstrin homology-like domain family A member 1) ist ein induzierbares zytoplasmatisches Protein, das einige Motive, die für die Vermittlung von Protein-Protein Interaktionen bekannt sind, enthält. Die PHLDA1 ist stark in gutartigen melanozytären Läsionen (Naevi) exprimiert und wird während der Tumorprogression des humanen Melanoms vom Primärtumor bis hin zur Metastase herunterreguliert. Das PHLDA1 Protein scheint als ein proapoptotisches Molekül zu fungieren. Das Mechanismus ist allerdings noch nicht bekannt. In dieser Arbeit wird gezeigt, dass die 293 PHLDA1 Transfektanten zusätzlich zu einer erhöhten Apoptose Empfindlichkeit auch eine höhere MHC Klasse I Oberflächenexpression im Vergleich zu Neo Kontrollzellen aufweisen. Das konnte mit mehreren monoklonalen Antikörpern, bestätigt werden. Auch mittels IEF, konnte auf der gesamten Proteinebene, bei zwei PHLDA1 Transfektanten eine höhere Expression der Allelprodukte HLA A2 und HLA B7 bestätigt werden. Nach einer 3-stündigen Inkubation mit radioaktivmarkierten Aminosäuren weisen die PHLDA1 Transfektanten 4,5- bis 8,5-fach mehr neu synthetisierte MHC Klasse I Moleküle, im Vergleich zu den Kontrollzellen, auf. Mit Hilfe der Pulse/Chase Methode konnte gezeigt werden, dass PHLDA1 Transfektanten, im Vergleich zu den Neo Kontrollzellen, einen schnelleren MHC Klasse I Transport vom ER zum Golgi Apparat aufweisen. Durch die Immunopräzipitation von MHC Klasse I Molekülen, konnte auch das PHLDA1 Protein mitpräzipitiert werden. Das PHLDA1 Protein war nicht mit ICAM-1 oder MCAM mitpräzipitiert, was eine spezifische Bindung des PHLDA1 Proteins an die MHC Klasse I bestätigt. Es ist denkbar, dass das PHLDA1 Protein als Chaperon fungiert und durch die Bindung an die MHC Klasse I Moleküle diesen eine höhere Stabilität verleiht. Dadurch können die MHC Klasse I Moleküle schneller an die Oberfläche transportiert werden. PHLDA1 Transfektanten wurden von HLA A2 allospezifischen T-Zellen besser als die Neo Kontrollzellen erkannt. So könnte der Verlust des PHLDA1 Proteins bei Melanomen und Mammakarzinomen auch zum Verlust der T-Zellerkennung beitragen.

Die adoptive T-Zelltherapie ist eine attraktive Alternative zu konventionellen Therapien zur Behandlung von malignen Erkrankungen. So konnten bereits Tumorremissionen bei Melanompatienten nach adoptivem T-Zelltransfer erreicht werden (Dudley et al, 2002b; Morgan et al, 2006). Während im autologen System jedoch oft nur unzureichende Antitumorantworten zu generieren sind, zeigt der Erfolg der allogenen Stammzelltransplantation, dass im allogenen System T-Zellen hoch effektiv Tumorzellen bekämpfen können. Die allogene Stammzelltransplantation konnte auch bei B-Zell-Non-Hodgkin-Lymphomen, wie beispielsweise der chronischen lymphatischen Leukämie (CLL), mit Hilfe eines Transplantat-gegen-Leukämie-Effektes (Graft-versus-Leukemia, GvL) lang andauerndes, krankheitsfreies Überleben bewirken. Sie birgt aber ein sehr hohes Morbiditäts- und Mortalitätsrisiko auf Grund der Transplantat-gegen-Wirts-Erkrankung (Graft-versus-Host-Disease, GvHD) in sich. Die im Transplantat enthaltenen T Zellen sind hierbei sowohl für den erwünschten GvL-Effekt verantwortlich, gleichzeitig aber auch für die unerwünschte GvHD (Horowitz et al, 1990; Kolb et al, 2004). Zur Minimierung des Risikos einer GvHD könnten T Zellen eingesetzt werden, die spezifisch und allorestringiert Peptide von tumorspezifischen Antigenen erkennen und somit bevorzugt Tumorzellen angreifen. Die Reaktivität der T Zellen kann durch einen T Zellrezeptor (TZR)-Transfer auf sekundäre Zellen übertragen werden. Diese transgenen Zellen können dann mittels adoptivem T Zelltransfer im Patienten zur selektiven Bekämpfung von Tumorzellen zum Einsatz kommen. In Vorarbeiten wurde FMNL1 (formin related protein in leukocytes 1) als hoch attraktives tumorassoziiertes Antigen identifiziert, das in der chronischen lymphatischen Leukämie (CLL) und in anderen Lymphomen, sowie in Zelllinien solider Tumoren stark überexprimiert wird, während es in gesunden Zellen fast ausschließlich in hämatopoetischen Zellen vorkommt. Ziel der vorliegenden Arbeit war es, allorestringierte FMNL1-peptidspezifische T-Zellen zu isolieren, zu charakterisieren und den T-Zellrezeptor dieser T-Zellen in sekundäre Zellen zu transduzieren. Hierzu wurden Peptide des tumorassoziierten Antigens FMNL1 mit Hilfe von Prädiktionsalgorithmen vorhergesagt und in T Zell-Stimulationsansätzen eingesetzt. Unter Einsatz von HLA-A2-positiven T2-Zellen als antigenpräsentierende Zellen, die mit dem prädizierten synthetischen Peptid FMNL1-PP2 beladen waren, ist es gelungen allorestringierte, FMNL1-PP2-spezifische T Zellen eines gesunden HLA-A2-negativen Spenders zu isolieren. Von 67 T-Zellklonen bzw. oligoklonalen T-Zellen konnte bei neun T-Zellklonen Allorestriktion und FMNL1-PP2-Peptidspezifität nachgewiesen werden. Der T-Zellklon SK22 war für diese neun T-Zellklone, die auf Sequenzebene einen identischen T-Zellrezeptor aufwiesen, repräsentativ. Der T-Zellklon SK22 zeigte in Reaktion auf peptidbeladene T2-Zellen eine hohe Peptidspezifität für FMNL1-PP2 im Kontext mit dem für SK22 allogenen HLA-A2. Nach Zielzellerkennung sezernierte der T-Zellklon Zytokine wie IFNγ, TNFα, GM-CSF und teilweise IL2. Der T Zellklon zeigte eine hohe Aktivität und mittlere Avidität gegen FMNL1 PP2-beladene T2-Zellen. Des Weiteren wurde die Reaktivität gegen unbeladene native Zellen getestet. Der T-Zellklon SK22 erkannte verschiedene Zellen, wenn sie HLA-A2-positiv waren und gleichzeitig FMNL1 exprimierten. Hierzu zählten zum einen maligne Zellen, darunter verschiedene Epstein-Barr-Virus (EBV)-positive und EBV-negative Lymphomzelllinien und die Nierenzellkarzinomzelllinie RCC26, die gut erkannt wurden sowie CD40-aktivierte CLL-Zellen, die schwächer erkannt wurden. Bei der Untersuchung von gesundem Gewebe wurden FMNL1-exprimierende HLA-A2-positive periphere Blutleukozyten (PBL) schwach und B-Zellen in mittlerer Stärke erkannt. HLA-A2-positive Zellen, die FMNL1 nicht exprimieren, wie beispielsweise Lungenfibroblasten, wurden vom T-Zellklon SK22 nicht erkannt. Der T Zellklon zeigte Kreuzreaktivität gegen neun verschiedene lymphoblastoide Zelllinien (LCL), die Allelvarianten von HLA-A2 exprimierten. Zusätzlich wurden 4 von 18 HLA-A2-negativen LCL-Zelllinien erkannt. Jeweils zwei dieser vom T Zellklon SK22 erkannten HLA-A2-negativen LCL-Zelllinien trugen ein gemeinsames MHC-Klasse-I-Molekül. Eines davon war HLA-A*3303, welches durch die Erkennung der HLA-A*3303-positiven Transfektante der C1R-Zelllinie bestätigt werden konnte. Das andere war HLA-A*6802, welches zur HLA-A2-Superfamilie gehört. Der T-Zellrezeptor des T-Zellklons SK22 wurde identifiziert, sequenziert und kloniert, sowie mit Hilfe von Retroviren in sekundäre Zellen eingebracht. Durch den Transfer des T Zellrezeptors von SK22 in sekundäre Zellen konnte nachgewiesen werden, dass dieser T Zellrezeptor für die spezifische Reaktivität des T-Zellklons SK22 verantwortlich war. Dies zeigte sich in der T-Zellrezeptor-Oberflächenexpression nach Transduktion in Jurkat76-CD8α-Zellen und in der Übertragung der Funktionalität des T-Zellklons in PBL. Der T Zellrezeptor von SK22 ist ein „schwacher“ Rezeptor, da er in der Konkurrenzsituation mit einem weiteren Rezeptor nur in geringem Grade an der Zelloberfläche von PBL exprimiert wurde. Durch einen Austausch der jeweiligen konstanten Regionen der T-Zellrezeptor-SK22-Sequenzen durch die konstanten Bereiche eines murinen T-Zellrezeptors konnten in der Summe verbesserte Expressionswerte in Jurkat76-Zellen und eine verbesserte Funktionalität in PBL erreicht werden. Der T-Zellklon SK22 zeigte Allorestriktion, FMNL1-PP2-Peptidspezifität und Zytotoxizität gegen FMNL1-exprimierende Zellen, insbesondere gegen Tumorzellen. Die beobachtete Kreuzreaktivität ist Fokus weiterführender Untersuchungen. Im Fall des T-Zellrezeptors von SK22 bedeutet es, dass Spender und Patienten sorgfältig nach Analyse des gesamten MHC-Klasse-I-Expressionsmuster ausgewählt werden müssen. Im Rahmen einer haploidentischen Stammzelltransplantation ist jedoch der klinische Einsatz dieses spezifischen T-Zellrezeptors zur Behandlung von B-Zell-Non-Hodgkin-Lymphomen und anderen FMNL1-überexprimierenden Tumorerkrankungen vielversprechend.

The goal of this study was to investigate the function of the heat shock protein 70 family members, expressed in tumors under physiological and stress conditions and to dissect their role in tumor immune recognition as a function of intra- versus extracellular location. Another goal was to investigate whether heat-treatment at clinically relevant thermal doses affects the immunophenotype of a given tumor, as defined by tumor cell sensitivity to immune effector cells. For these questions, the human melanoma system was selected because it is well characterized with regards to tumor-associated antigens, like tyrosinase and Melan-A/MART-1, their epitopes and restriction elements for MHC class I presentation. In the first part of the study the focus specifically was on the time-temperature dependent effects of heat exposure. Two different thermal doses (41,8°C/120 minutes and 45°C/22 minutes) were selected that mimic the heterogeneity of the achieved temperature distribution within the tumor and the time-temperature dependent changes were determined in: a) antigen expression (tyrosinase and Melan-A/MART-1) at the protein and mRNA level; b) expression of the inducible HSP70 and the constitutive HSC70; c) processing and presentation of tyrosinase and MART-1 via MHC class I; d) susceptibility of melanoma cell lines to cytotoxic T lymphocytes like CD8+ T cells, LAK and NK cells. It was demonstrated that HSP70 and antigen expression display distinct expression and kinetics that reflect the thermohistory of the cells, i.e. exposure to high or low thermal doses. Immunologically, a low thermal dose did not alter immune recognition of the cells despite the fact that intracellular HSP70 and tyrosinase protein were upregulated. High thermal dose induced a pleiotropy of effects, including stronger upregulation of HSP70 and tyrosinase protein but downregulation of tyrosinase at mRNA level. Concordant with reduced HLA-A2 surface expression and tyrosinase mRNA levels, immune recognition of the heat-treated cells was initially reduced, but pretreatment levels were restored after 72 hours of recovery. The observation that tumor cells treated with temperatures below the breakpoint temperature maintain an immunological homeostasis during the heat shock response is of critical importance for the clinical application of hyperthermia in the treatment of tumors. In the second part of the study, the ability of HSP70 to cross-present a naturally expressed human tumor antigen, tyrosinase, that is of low immunigenicity, a situation that more closely resembles the patient situation was investigated. It was demonstrated that HSP70-peptide complexes (HSP70-PC) purified from tyrosinase-positive (HSP70-PC/tyr+) but not from tyrosinase-negative (HSP70-PC/tyr-) melanoma cells deliver the tyrosinase antigen to immature DCs for MHC class I restricted T cell recognition. T cell stimulation by HSP70-PC/tyr+ incubated with immature DCs with was very efficient even without additional DC maturation signals (e.g. exogenous TNF-?) demonstrating the ability of tumor-derived HSP70-PC to act as a chaperone for peptides and a signal for DC maturation. HSP70-PC in exerting both functions on DCs, delivering antigens and maturing DCs, ensures that the peptides that are delivered to the DCs are presented in an immunogenic context optimal for T cell stimulation. In conlusion, induction of intracellular heat shock proteins (HSPs) by heat does not interfere with the tumor immune recognition and when HSPs are expressed extracellularly they acquire immunostimulatory properties. These observations open new perspectives for the application of hyperthermia in combination with HSP-based vaccine in the treatment of solid tumors.

Computer simulation of the conformations of short antigenic peptides (&lo residues) either free or bound to their receptor, the major histocompatibility complex (MHC)- encoded glycoprotein H-2 Ld, was employed to explain experimentally determined differences in the antigenic activities within a set of related peptides. Starting for each sequence from the most probable conformations disclosed by a pattern-recognition technique, several energyminimized structures were subjected to molecular dynamics simulations (MD) either in vacuo or solvated by water molecules. Notably, antigenic potencies were found to correlate to the peptides propensity to form and maintain an overall a-helical conformation through regular i,i + 4 hydrogen bonds. Accordingly, less active or inactive peptides showed a strong tendency to form i,i+3 hydrogen bonds at their Nterminal end. Experimental data documented that the C-terminal residue is critical for interaction of the peptide with H-2 Ld. This finding could be satisfactorily explained by a 3-D Q.S.A.R. analysis postulating interactions between ligand and receptor by hydrophobic forces. A 3-D model is proposed for the complex between a high-affinity nonapeptide and the H- 2 Ld receptor. First, the H-2 Ld molecule was built from X-ray coordinates of two homologous proteins: HLA-A2 and HLA-Aw68, energyminimized and studied by MD simulations. With HLA-A2 as template, the only realistic simulation was achieved for a solvated model with minor deviations of the MD mean structure from the X-ray conformation. Water simulation of the H-2 Ld protein in complex with the antigenic nonapeptide was then achieved with the template- derived optimal parameters. The bound peptide retains mainly its a-helical conformation and binds to hydrophobic residues of H-2 Ld that correspond to highly polymorphic positions of MHC proteins. The orientation of the nonapeptide in the binding cleft is in accordance with the experimentally determined distribution of its MHC receptor-binding residues (agretope residues). Thus, computer simulation was successfully employed to explain functional data and predicts a-helical conformation for the bound peptide

Antigen HLA-B27 is a high-risk genetic factor with respect to a group of rheumatoid disorders, especially ankylosing spondylitis. A cDNA library was constructed from an autozygous B-cell line expressing HLA-B27, HLA-Cw1, and the previously cloned HLA-A2 antigen. Clones detected with an HLA probe' were isolated and sorted into homology groups by differential hybridization and restriction maps. Nucleotide sequencing allowed the unambiguous assignment of cDNAs to HL4-A, -B, and -C loci. The HLA-B27 mRNA has the structural features and the codon variability typical of an HLA class I transcript but it specifies two uncommon amino acid replacements: a cysteine in position 67 and a serine in position 131. The latter substitution may have functional consequences, because it occurs in a conserved region and at a position invariably occupied by a species-specific arginine in humans and lysine in mice. The availability of the complete sequence of HLA-B27 and of the partial sequence of HLA-Cw1 allows the recognition of locus-specific sequence markers, particularly, but not exclusively, in the transmembrane and cytoplasmic domains.