 What I want to do is look at, give an overview of the tumor microenvironment kidney cancer because we think obviously that this environment can lead to immune dysregulation and if we can understand more about the tumor microenvironment, we might be able to come up with additional targets for immunotherapy. So these are my disclosures. And so just talked about the tumor microenvironment. The tumor is not just the tumor cells, but in fact there's a number of different kinds of cells, fibroblast, myeloid cells, various immune cells, but turns out that many of these cells that are infiltrating the tumor are dysregulated, functionally impaired and contribute to the tumor progression. And this is illustrated here. This is, I took this from Nick Rostevo and you can see that there's the tumor microenvironment here. And these are the tumor cells and these are the infiltrating cells. So it was originally thought that these cells were all coming in to promote an anti-tumor response, but it turns out many of these cells are there to promote the tumor. So it's the opposite of what we originally thought. And so the idea now is to try to understand this better. And this is occurring now with the interactions, looking at PD1 and PDO1. So this is just a slide from Walt Storkas to showing that in kidney cancer patients in melanomas there is a decrease in the type 1 gaminephuron response and it's linked to active disease. So the only time you see in these patients a T-cell CD8 response was when they either had no evidence of disease or they had disease, but they were long-term survivors. And this also shows that these cells are skewed towards the type 2 gaminephuron, type 2 response rather than gaminephuron. So they're making more like immunosuppressive molecules like IL5 and 4. So they're all making IL5, but they're not making gaminephuron with the exception of the ones with NED. So people that have, so in this setting the tumor is actually regulating the development of a T-cell response. And we've seen this in kidney cancer and in melanomas. So what are the features that can promote this dysfunction? Well it's, you can have a level of T-cell priming where you have function, reduced function of dendritic cells, reduced, reduced the recruitment of these cells into the tumor. You can get less recruitment of effector cells because maybe the chemokines that we're using to attract them are not there. There's also, as we're going to talk about, there's presence of these immunosuppressive cell types. I'm not going to talk about the PD-01 and CTL-4 because that's being covered. So I'm going to focus a bit on these cell types. So as you know there's T-regulatory cells that have the FoxB3 that helps regulate the suppressive activity of these cells and these cells can come from the thymus and go into the tumors and where they can suppress. You can also take naive T-cells and after exposure to cholerogenic DCs, the presence of TGF beta, they can become T-regulatory-like and suppress. And you can see that there are multiple mechanisms by which they can suppress immune response. It's looking now like CTL-4 may be one of the more important components here. But there are multiple mechanisms. And if you look at, there's always, in most of these cancer patients, this is kidney cancer, there is an increase. It's a modest but an increase in these T-regs within the blood and you can also see them in the tumor and in some cases I think we looked through the literature and there was nine papers showing that there's an increase in T-regs in the patients with kidney cancer but only four of those related to poor clinical outcome. So there's a little more work to be done to look at that, how this relates to outcome. You can also show that in this paper that if you deplete the T-regs by using IL2 that's has a diphtheria toxin, you can improve the T-cell response to the vaccine as shown here. So the other cell that's important and that's the one we've been studying more is the myeloid drive suppressor cells, heterogeneous population of myeloid cells that are present at less than 1% in the peripheral blood of normals but are increased in response to a variety of different growth factors as shown here. These cells can leave two flavors, they're either granulocytic or monocytic and mice and the same populations here but in humans there's also a lineage negative population and these cells are interesting because they have a lot of plasticity. They can differentiate into these endothelial cells or they can differentiate into macrophages or the monocytes can differentiate into granulocytic cells. So understanding that process I think is going to be important. So in many cancers including kidney cancer, lung cancer, brain cancer, the largest population of the granulocytic MDSCs and that's shown here, these guys, and that there's now some data suggesting from a paper recent in Nature Medicine that the levels of these does correlate with bad outcomes. So patients that had baseline levels of low levels of these MDSCs, the monocytic population had a better survival curve than those that had higher levels and this has also been reported in the same paper for the granulocytic population and there's also data on several other tumor types. So it's been shown that these cells are immunosuppressive so here they are suppressing T-cell proliferation and you can dilute them out and there's less of them, there's less suppression. We also find that these cells probably promote angiogenesis because if you inject these tumors into nodskin mice, these are human tumors, you can see the blood vessels after five days but if you put in a few of these granulocytic MDSCs, you can increase the vascular component here of these tumors so they also make pro angiogenica molecules. So how do they suppress, well they do so by different mechanisms. One of the major ones is they produce arginase and nitric oxide where they can then down-regulate or reduce the amount of these two amino acids which then causes T-cell dysfunction and you have the loss of the zeta chain. They also can produce reactive oxygen species and they can also make nitric oxide which can then nitrate the T-cell receptors and cause dysfunction. Indirectly they can induce T-regs, cells they can induce TH-2s and macrophages. Nutri-fills we also think are important, they're just being studied and there is elevated level of these in kidney cancer and other patients that correlate with bad outcome and if you look at the neutri-fills from these patients, they are, when you compare them to normals, suppressive as shown here and these cells can also promote, unlike normal neutri-fills which don't, can promote the vasculature when you mix the tumor with these granulocidic imidazes and you put them in the not skid mice. So we do think they play a role as well and that's being studied. Macrophages can also be part of the problem here because under normal tissues you have these macrophages which can promote a type I inflammation, a type I response and a tumor acetyl. But in the tumor maleu you have a bunch of cytokines and other growth factors and products that can shift these to this kind of a phenotype and there could be different levels of this M2 phenotype and it's produced primarily by exposure to IL-4, IL-10 and 13 and this then drives these cells to suppress T-cell function, suppress the type II, type I response and they can promote immune suppression. This is just one example in kidney cancer, it's recently identified that there's a number of MDTAMs in the tumor and they have an up-regulation or activation of the lipoxygenase pathway leading to the production of these lipids and these lipids can either promote more monocytic infiltrates, reduce IL-10 and also promote the induction of these T regulatory cells. And also, because there's this kidney cancer where you have the VHL mutation, you have constitutive Hifone Alpha and you have hypoxia, that also can promote immune suppression, you can see here the T-regs are increased in this setting, there's increase in the chemokin that can promote these cells into the tumor, it also can induce more MDSCs to be more suppressive and also to differentiate into tumor-associated macrophages and on the flip side it can down-regulate dendritic cell ability to present antigen. So this tumor buyer is pretty hostile for the immune system. Just the one slide I have for the PD-01 is that there's renal tumors, lines that have PD-01 and if you can down-regulate that on the surface with the SIRNA you can see that there's some reduction in the ability of these tumors to induce apoptosis in T cells and you'll hear more about the fact that PD-01 is expressed in kidney cancer to correlate with poor outcome and it's not only expressed on the tumors but on macrophages and dendritic cells and there's also a secreted form of this. This is just a slide to show that one of the important equations here are the chemokines and chemokine receptors which we're going to study as well as others and this just shows the kinds of receptors are on these different suppressive cells and here are the ligands that combine and attract these cells into the tumor. So which of these is the most important for trafficking is a good area of investigation going on. There's a lot now that just made a short list of all the different strategies used to reduce these MDSCs or TAMs or T-regs and so there are now different methodologies you can use to reduce these and so it'll be interesting to see what effect this has when you combine this with vaccines and with other approaches. I just wanted to mention one example of modulating the tumor microenvironment with synitinib. So we have shown that in cancer patients, kidney cancer, you can see a reduction in the number of MDSCs over time and that's their corresponding increase in T-H1 response came in if you're on and if you look in the tumor it's more of a mixed bag where some of these papers had very low levels of MDSCs and good T-cell game interferon response but the others had retained the MDSCs and there's no T-cell response and so we have some now data that suggests that these myeloid cells are involved in the resistance to some of these TKIs and there's another group that has similar data. So because they can suppress MDSC numbers and T-regs and can improve T-cell game interferon response, people have combined this with the various vaccines and I think there's like four or five papers now on this and this is one by Anna Mikobos and Walter Sorkes' lab where it shows that this combination can reduce the tumor volume better than either drug alone but this slide actually shows that it can really improve the T-cell response. You get a great increase in CD8s, CD4s, the loss of MDSCs and the loss of T-regulatory cells and there's also in the tumor if you look there's a skewing towards a type 1 response both in terms of game interferon production as well as in game interferon production and the cytokines and receptors that are associated with the type 1 response and based on that there is now several, two clinical trials ongoing to compare to look at whether or not synitinib will be advocated with the vaccine therapy and we're beginning to look at another vaccine with Walter Sorkes looking at the targeting antigens on the tumor vasculature. This is just to show that not only do these TKIs working with vaccines but there are people now that are looking at synitinib and sopimid with anti-PD1 and probably CTLA4 so that's coming down the line. So in conclusion there is heterogeneity in patients response to immune-based immunotherapy and it's a significant component of that heterogeneity comes from differences at the level of the tumor microenvironment. So the key determining factors in the tumor environment include the recruitment of the factor T cells, local production of cytokines and the presence of local immunosuppressive mechanisms. So further understanding these processes we think will uncover additional targets and it's pretty impressive to see what the studies have shown so far that CTLA4 and PD1 have been major players now. So I think I will stop here. This is usually sunny days in Cleveland so.