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HuMouse models for the influence of genetic variation on the immune control of Epstein Barr

Christian Münz, Ph.D. - Professor and Co-director of the Institute of Experimental Immunology, University of Zürich, Switzerland

Talk Title: Humanized mouse models for the influence of genetic variation on the immune control of the Epstein Barr virus


Welcome to this journal club. So in the next 20 minutes, I will try to tell you a little bit about a study that we recently concluded, where we tried to use the genetic variability in the hematopoietic progenitor cells that we use for humanization in our humanized mice, to learn which genetic factors might influence the infection by the common human pathogen Epstein-Barr virus. Which at the same time, as it is a very common pathogen in humans or persistently infects more than 95% of the human adult population, the same time. It's also one of the most gross transforming viruses that we know, a virus that we can actually use as most of you have maybe done at one time point to another, to immortalize B cells in cell culture. And we would like to exploit a genetic variation to learn more about this immune control. So why is that maybe of general interest to study Epstein-Barr virus? It is a pathogen that in addition to the fact that it causes or is associated with tumors, it is also a pathogen that is explicitly immune controlled by cell-mediated immune control.

What I mean with that is that a primary immune deficiency. So genetic variations in humans that predispose for EBV associated diseases, so basically convert and asymptomatic carrier state of EBV to pathogenesis. Those prime immune deficiencies with genetic lesions in individual genes all mark cytotoxic lymphocytes here shown in this schematic graph. And cytotoxic lymphocytes, primarily CD8+ T cells. So cytotoxic T cells as important to control EBV infected B cells. And these primary immune deficiencies mark signaling molecules in the T cell receptor itself in co-stimulatory molecules that are required for the recognition of EBV infected B cells and the cytotoxic machinery that is used for the cytotoxic lymphocytes to kill EBV infected cells. We would like to use EBV in humanized mice to learn more about what requirements are there to control a potentially very dangerous human pathogen, that can cause tumors such as Hodgkin's lymphoma and Burkitt's lymphoma, lie long in most of us without causing such pathologies.

Why did we actually particularly focus our attention on MHC class II haplotype that is present in the human population? Namely, the MHC class II molecule, HLA DRB1*1501, and the MHC haplotype that is associated with MHC class II molecule. The reason why we focused on that is because this particular MHC class II molecule is tightly associated with genetic risk to multiple sclerosis, multiple sclerosis being an Auto immune disease of the central nervous system in which presumably CD4 T cells are causative in this disease and are restricted by DRB1*1501, which I will abbreviate just as DR15 in the rest of the talk. So this particular MHC class II molecule, DR15, that increases the risk to develop MS about threefold, but it was known fairly early on that this is not sufficient. So the genetic risk is not sufficient to develop the disease cause motorcycle wreck twins actually have only devious risk to basically get both the disease of 20 to 30%.

So in addition to the genetic background, the environment modify this risk. And as you can see in this table from a recent value of two months or worse in school, too much, Austin heading the Scandinavian domestic registry out of the Colin's kind of attitude, you can see that a number of environmental risk factors have been identified over time. And in this particular, in this particular table, you can actually see that some of these environmental risks factors listed on the left interact with this MHC class II molecule to further increase the risk and among these risk factors are altered Epstein Barr virus specific immune responses, particularly antibody responses against the Epstein-Barr virus and also, and symptomatic primary infection of Epstein-Barr virus called infectious mononucleosis. And this brings up the risk to develop MS from this threefold that you see in a particular immune class II background, to 15 fold in the case of an elevated antibody response to Epstein-Barr virus and to seven fold after infectious mononucleosis. And then all the time has now emerged. Actually maybe the strongest environmental risk factors that is synergistic with HLA-DR15 and development of multiple sclerosis.

There are also investigators that even more strongly believes that EBV infection has to always proceed MS onset one of these investigators is Alberto Ascherio, from Harvard, he has studied the military personnel cohort and the American nurses' cohort. In these two cohorts, which are sampled regularly in peripheral blood, and then different disease manifestations can be investigated longitudinally. He has shown that EBV infection always precedes MS by usually a couple of years. And he has not been able to identify any individual that was Epstein-Barr virus negative and develop multiple sclerosis. But the strongest evidence I would personally say is probably the, then epidemiological evidence that EBV or certain types of EBV infections synergize with HLA-DR15 to develop an alleles.

In addition to this MHC class II molecule HLA-DR15 over time, more than 200 genetic risk factors or single nucleotide polymorphisms in the human genome have been identified the modulator MS with, but even zone these more than 200 snips have now been identified in these studies that are the basis for this identification usually involve multiple 10,000 individuals and patients and the multicenter cohorts until idea 15 is still the strongest respect from MS with an odds ratio of 3 while most of these other co-stimulatory molecules on cytokines that mostly at home to the CD4 helper T cell lineage. These only have odds ratios between 1.1 and 1.5. So modify the risk rather than mightly in comparison to the MHC class II molecule that is associated with MS.

Now a little bit, the rationale why we wanted to investigate this question. So in our humanized mouse model, to which extent, HLA-DR15. HLA-DR15 brought in by the hematopoietic progenitor cells that we use for a constitution of in this study, we have used Netscape common gamma chain, knockout mice, mostly without any HLA trans-gene. If these immune compartments that reconstitute over three months would then behave differently to Epstein-Barr virus infection, from the Epstein-Barr virus infection that we use, users between 10 to the three and 10 to the five infectious virus particles, the data that I will show you mostly from this 10 to the five higher dose of infection, which actually mimics a sort of symptomatic primary infection like infectious mononucleosis in humanized mice with a quite significant expansion of CDA T cells and quite high amounts of cytokines, pro-inflammatory cytokines that are secreted. You can use the media infected humanized mice to analyze viral loads, the tumor formation, following Epstein-Barr virus infection, and also how the immune compartments react to them.

So if you had been done, doing these types of experiments for quite some years, more than 10 years, and what we have noted in most of our experiments quite consistently, and you might be familiar with similar reconstitution plots, that when we look after three months in the peripheral blood of humanized NSG mice, then we find the lymphocyte gate, quite an abundance of human cells as identified by human CD45 staining. Within these human lymphocytes, the majority of the cell that this three month time point around 60% is still B cells while around 30% are T cells. Within the T cells, we have an abundance of CD4 helper T cells, and a minority of cytotoxic CDA T cells. So around between 20 and 30%, miss some heterogeneity in the reconstitution. You also find in the lymphocyte gate, around 2% of the NK cells, mark band, KP 46. And this was from a recent study that is about to come out.

You also have now recently established a tool in the laboratory to look at this very constitute histologically called chip cytometry. You can see a spleen section of a humanized mouse, and you might be familiar with this picture, that after reconstitution, we see that there are white polyp areas that have formed in this humanized mouse plane around venules and arteries, but the white polyp areas, even though they have some degree of segregation between CD20 stain D cells and maybe more peripherally CD8 T cells, they don't form or don't show any clear germinal center automation. But you can use this chip cytometry, which are basically repeated, staining bleaching cycles to gain knowledge about for now about 40 markers in these tissue sections to localize different T cell compartments, different co-stimulatory molecules on these T cells in the reconstituted mice.

Well, the fact that we have done these reconstitutions for quite a while. So when we started our HLA-DR15 project, we actually look back at all the reconstitutions that we had done over time after HLA typing CD34 hematopoietic progenitor cells, and just pooled all the data to look for T cell compartments, CD4 and CD8 T cell compartments that had to be reconstituted in these mice. And these will present several thousand mice derived from more than 200 donors. And what we noticed, surprisingly, even though you would think that across so many different reconstitutions, and to some extent also different investigators, you will find no difference. When you look at a particular image C class two molecule, here, we compare HLA-DR15 negative, and HLA-DR15 positive reconstitution. We found to our surprise, we find a higher activation of T cells. And that is primarily in the CD4 T cell compartment, which then is reflected in the overall T cell compartment on higher activation in the context of DR15.

And you use here MHC class II expressions or HLA-DR expression as an activation marker for these T cells. Then we're wondering if that is actually, can be compared to other MHC class II molecules, and as a comparative used here, HLA-DR4, and you have your 1041, which is an emergency class II molecule that is associated genetically with other auto immune diseases, diabetes, and also rheumatoid arthritis. But we did not see that HLA-DR4 reconstituted mice in higher T cell activation comparison is HLA-DR15 reconstituted mice. We still saw a higher activation of T cells in the DR15 reconstitute mice, suggesting the HLA-DR15, for some reason, triggers a higher activation on the reconstituted T cell compartment in humanized mice, after three months.

You also looked, and that's why I showed you a little bit, this, this slide in the beginning on the more than 200 snips that are associated with MS. We looked at also at 200 of these single nucleotide polymorphisms that are associated with them MS in addition to the MHC class II molecule, and these snips, we had to type, so we type them in a subset of donors. So 21 donors as you can see, and you can see why there was actually quite a significant increase in activation with HLA-DR15, the difference in accumulation of snips that are associated with Multiple Sclerosis in humanized mice resulting in a higher activation of the T cell compartment that is now not yet convincing.

And also this increased snips load is not statistically significant, even though for some individual snips. And here a snip is shown in EOMES transcription factor that is often associated with cyto toxicity in the lymphocyte compartment. There is a tendency, or even in this case, statistically, significant of an increase T cell activation in the presence of this snip. But because the snips probably do not confer a very strong alteration in the immune response, and that is also then not translate or kind of be translating a much higher risk to multiple sclerosis. Also, our humanized mouse system probably does not allow with a small amount of animals to actually see a significant increase in T cell activation. But that's a little bit, I guess, the limitations of the system, that snips that have a mild effect on the immune compartments, but it probably would not show up as a significant in this humanized mouse system.

We then went further and characterized the HLA-DR15 reconstituted mice. And what we found is that after EBV infection, so using these 10 to the five infectious viral particles of EBV, we see that in the HLA-DR15 reconstituted mice, T cell expansion is increased. These show total numbers of CD4 and CD8 T cells even peripheral blood or, or spleen. And you can see a higher expansion of CD4 T cells, and also CD8 T cells after EBV infection.

Okay, despite this higher expansion of T cells, we find that in the HLA-DR15 positive animals, viral loads of Epstein-Barr virus shown here as EBV copies in the blood are increased. And again, when we stratify towards HLA-DR4 reconstituted, with MHC class II, haplotype in HLA-DR15 reconstituted animals. You see that also still holds true, and you can see that the DR4 actually are not distinguishable from the bulk of HLA-DR15 reconstituted animals.

Sorry for that, clicked too far. So then we then looked at correlations between the EBV copy numbers in the spleen and the T cell expansion. In this case, CD8 T cell expansion, we see that similar to infectious mononucleosis. So this primary infection, symptomatic infection is EBV. You find a correlation between the T cell expansion and the viral loads suggesting that indeed this maybe sub optimal immune control of EBV, allowing for higher viral loads is associated with stronger CD8 T cell expansion, suggesting that the CD8 T cells are driven by this antigenic load, but are not capable of controlling EBV as well as another MHC class II haplotypes. And you can also observe in a subset of mice IgM responses to a particular antigen called the Epstein-Barr virus, nuclear antigen number one, and also these antibody responses in the IgM compartment, somewhat correlate in the subset of mice, where they can be actually detected with the B viral loads.

Then we looked a little bit in more detail at, if source massively expanded T cell compartment might actually show some evidence for directly recognizing auto antigens that have been associated with multiple sclerosis. And we had done something similar more than 10 years ago in MS patients, where we looked actually at Aetna one specific T cell clones, Aetna one specific CD4 T cell clones, to which extent they would cross react with MS autoantigen. So these were peptides from myelin basic protein, oligodendrocytes protein and PLP. And we found at the time that when you compare it to other autoantigen for insulin from diabetes, and you can find that a subset of clones actually recognize as both Aetna one and myelin antigen. So had some crossway activity against auto antigens.

And because we had this prior experience, we also look then in collaboration with what I'm marking here from the neurology department that studies a lot T cell responses in MS patients with a peptide mixture, again of different MS associated auto antigens. If you could find some evidence in EBV infected humanized mice in the context of HLA-DR15 for recognition of these older antigens, and this recognition is monitored by ELISpot. So spot forming cells in 10,000 splenocytes of these infected mice. And you can see that there are some, even though it's a smaller signal, there are some signal that MBP myelin basic protein is recognized in the splenocytes of humanized mice that have been EBV infected in the context of DR15. Why this picture is more heterogeneous in the context of DR4. And therefore there might be some cross-reactive mice, but the larger amount shows actually no cross reactivity

Then cloned these T cells from splenocytes of humanized mice and the system that we used to focus the clones on the particular image C class II molecules that we were mainly interested in is that we used EBV transformed B cell lines from bare lymphocyte syndrome patients.

So these are patients that do to genetic variability have no MHC class II on their surface. And these lymphocytes in them LCL were then transfected either with HLA-DR15 or HLA-DR4. So there were a single MHC class II expressing, and then we stimulated splenocytes from the respective humanized mice. These are DR15 reconstituted or the DR4 reconstituted after EBV infection with these bare lymphocyte syndrome LCLs and by virtue of interferon gamma production towards if LCLs we pulled our clones. Here you can see then the readouts of these clones that we derived. And we asked the clones to recognize by sight of toxicity, either DR15 positive LCLs or the DR15 negative LCLs and vice versa for DR4 autologous or allogeneic LCLs. And here the killing assay for six hours, heres the killing assay for 18 hours. And you can see that the clones that we derived, they're capable to recognizing through DR15 or DR4 quite efficiently, maybe transformed cell, cells and killed them to a similar extent.

However, when we look at cytokine production, then production of interferon gamma, production of TNF alpha, and IL2 was very specific for the DR4 restricted clones while the DR15 restricted clones produced, to some extent the cytokines also quite significantly to allogeneic targets suggesting that indeed there may be a broader specificity that they can make use of more than their own, their own restricted image T class II molecule. And this broader activity could explain in part, an inflammatory response that even extends that I've shown you to some extent to auto antigens of MS.

We'd like to summarize this study that I presented to you. So we believe that T cell compartments are hyper activated in the context of HLA-DR15 and around the same time as we studied this in our humanized mice, our collaborator Ole Martin has studied that in vitro. And it has shown that when you actually take these cells and stimulate T cells in the context of HLA-DR15 and he observes and autologous MLRs or stimulation of the T cells by the B cells without any additional antigens.

So it seems like that the T cell compartments are somewhat driven by HLA-DR15 positive B cells in an autologous setting. And this reactivity is enriched in MS patients and might be connected to this particular MHC class II molecule. You found that these T cell compartments that are already in the steady state, hyper activated, they expand to higher levels in response also to EBV infection, despite this elevator expansion, they control EBV infection less well. Curiously even though both C4, the C4, I believe that T cell, that are restricted by HLA-DR15. You also found that CD8 T cells expanded to a large extent, but the things that might be secondary to the increased viral loads, they correlate also maybe your line loads. And the actual idea of 15 was tactically for T cell clones, that we were able to derive theme to have a, a quite significant, and also quite selective. So of toxicity against EBV infected B cells, but this play cross reactivity with respect to from, from tourist cytokine production.

So the open questions obviously are, if indeed, in this particular MHC class II contexts infection EBV infection is less controlled. Where would such EBV infected antigen presenting cell would be located? Is there an inflammation caused by these T cells that might be stimulated by these less well controlled B cells and therefore be associated with MS pathogenesis? So I could think of such a risk stimulation maybe in the central nervous system and how can I induce EBV specific immune control be strengthened in this particular HLA haplotype to risk/reward immune controls.

So to put it in a cartoon. What we think could be one component of the devious process in MS and that has also been put forward by Alberto Ascherio in this review in 2012, is that indeed less well controlled EBV infected B cells might be able to migrate to the CNS, to the brain. And there, locally re-stimulate B cells that either by virtue of their pro-inflammatory cytokine production alone or by recognition of autoantigens myelin derived auto antigens contribute to the multiple skills.

We would just like to finish by thanking the people that ran, worked in this work. So it was a study that was published in the European journal of immunology, was spearheaded by a PhD student, Hana Zdimerova with help from Roland Martin, our collaborator at neurology partners, Zurich University Hospital, and a postdoc who was at the time in my lab and has now moved to Cell Medica, Bithi Chatterjee, and Innosuisse funding agency and you for your attention and take any questions that you might have.