Right. Thank you so much. My name is Burkett Heiles-Lasse. I am from here, from Stanford and Lucille Packard Children's. I'm very grateful to be presenting the Year in Review for Basic and Translational Research. So I have no particular disclosures that are pertaining to the topics we'll highlight today. And what I would like to discuss are a couple of publications that have come out over the last year that perhaps give us some novel insight for our practice and may augment our clinical management in the near future. And these include a novel role for red blood cells as immune sensors in critical illness, followed by immune modulatory effects of recently approved angiotensin II, agonist geopressa, and preclinical models of sepsis. And then we'll close off by looking at cell-based therapies for pediatric diffuse middle angiomas, some of the preclinical findings in the phase one trial that's ongoing currently. So the first paper that I want to highlight was published in Science Translational Medicine, and it looks at the role of red blood cells as immune sensors. And so generally, when we think about red blood cells, we think about them as key mediators of gas exchange, nitric oxide bioavailability, and often they're thought of as immunologically inert. However, we know that they do carry and absorb cytokines and chemokines, therefore indirectly modulating the immune response. And anemia is a very common complication of both sterile and non-sterile inflammatory insults. So the investigators wanted to look at whether or not red blood cells do express pattern recognition receptors that respond to danger signals and subsequently modulate immune response. And what they've shown in this publication is that red blood cells indeed do have pattern recognition receptors, in particular, TLR9 receptors on cell surface of the red blood cells. And they show that they're immunologically active. Generally, TLR9 receptors pick up unmethylated CpG motifs of either bacterial or mitochondrial DNA. And what they do in this experiment is do a co-culture experiment using culture media from plasmodium phosphorum and incubating it with red blood cells. And they show in a dose-dependent manner that the plasmodium DNA is picked up on the surface of these red blood cells. They go on to ask whether or not this has impacts on red blood cell morphology, also red blood cell function. And they do that by using synthetic single-stranded DNA that has the unmethylated CpG motif. And so they incubate it with red blood cells in vitro and show with these electron micrographs that the amount of alteration in red blood cells has significantly increased. And also, the cell surface markers that are important for survival of these red blood cells, for example, CD47, is downregulated. And so naturally, the next question they asked was whether or not this augments hemophagocytosis as well as an inflammatory response. And they do this by using the same unmethylated CpG motif that's synthetic and incubating it with red blood cells that have a GFP tag to them, and then subsequently transfusing it to wild-type mice and looking at splenic sequestration as well as pro-inflammatory response. And what they show here is when you transfuse red blood cells that have, in theory, bacterial and mitochondrial DNA loaded on them, they do have an increase in splenic sequestration by splenic weight as well as an increase in hemophagocytosis that's seen by the amount of GFP signal that's seen in splenic macrophages. And they also show that it does induce a pro-inflammatory response, in particular, interferon gamma as well as IL-6 response. And ultimately, they bring it back to the bedside and validate their preclinical findings by showing that in a cohort of septic patients as well as ARDS, that the TLR9 signal that's found on the cell surface of red blood cells is upregulated. It also loads higher amounts of mitochondrial DNA. And they show that, in particular, their severe ARDS patients, there's an inverse correlation between the amount of mitochondrial DNA that's loaded on red blood cells and the hemoglobin concentration. So I believe this paper is quite important for us because not only does it give us a new role for red blood cells as immune sensors and a critical illness and gives us insight on the pathogenesis of anemia and chronic infectious and non-infectious inflammatory diseases, but also I think it would have implications for transfusion reactions as well as complications of solid or liquid transplant that we need to think about in the near future. And also provides us new therapeutic targets for immune modulation that require future investigation. The next paper I want to highlight looks at angiotensin II and its immune response or immune modulatory effects in preclinical sepsis models. And this was published in PNAS. And the premise is focused on the fact that catecholaminergic vasopressors like epinephrine and norepinephrine have been associated with immune suppression, both in preclinical sepsis models as well as in clinical observational studies. And recently, with the conclusion of the ATHOS-3 trial, angiotensin II or Giapreza has been FDA approved for treatment of vasodilatory shock in adults. And while we know that angiotensin receptors are present in a variety of cell surfaces, including innate immune cells, the impact of angiotensin II on innate immune function, particularly in sepsis, is really not well delineated. And so that's what the authors tried to look at in this publication. And what they did using a sequel ligation puncture model is first they looked at changes in angiotensin receptors on innate immune cell surfaces following CLP. And what they show is that, in particular, angiotensin type 1 receptor is upregulated following sequel ligation puncture, both in inflammatory monocytes and neutrophils from the spleen and peritoneum, suggesting that if angiotensin was going to or angiotensin II is going to modulate immune response, it perhaps would be through this specific receptor. And so they test the hypothesis by treating mice that have gone through sequel ligation puncture with either angiotensin II or angiotensin plus Losartan, which is an angiotensin type 1 receptor inhibitor, and then subsequently look at innate immune function using ex vivo LPS-induced phagocytosis capacity as well as ROS production. And what they show is that following sequel ligation puncture, treatment with angiotensin II seems to augment the innate immune response in this, especially in neutrophils and inflammatory monocytes, by increasing LPS-induced phagocytosis capacity. And this effect seems to go away when you co-treat with Losartan, suggesting that this mechanism is through the angiotensin type 1 receptor. They go on to show that it has implications for bacterial clearance. And so this is the same model, same treatment conditions. And they look at bacterial burden in the peritoneum as well as in blood and show that when you treat with angiotensin II in a sequel ligation puncture mice, the bacterial burden both in circulation and the peritoneum is significantly decreased. And that's reversed also with Losartan. So in summary, this study shows that angiotensin II seems to augment innate immune response in comparison to catecholaminergic vasopressors, which seem to suppress. And this seems to be mediated through the angiotensin type 1 receptor found in the cell surface of myeloid cells. And this gives us some mechanistic insight into some of the subgroup analysis that was seen in the ATHOS-3 trial, which suggests survival benefit with angiotensin II in a particularly severely ill subgroup of their patients. And then it sets up the foundation for future clinical studies, which evaluate the immune modulatory effects of angiotensin II and subsystem types, particular subsystem-induced immunoparalysis. And the last two papers I want to highlight deal with the cell-based therapies for diffused midline gliomas, in particular diffuse anti-GD2 CAR-T cell therapies. Diffused midline gliomas, which include DIPG, are high-grade midline tumors of the brain stem, as well as spinal cord. They account for majority of the pediatric brain stem tumors, and they're historically very aggressive. About average lifespan post-diagnosis is about 10 months. Less than 1% survive five years. And currently, the therapies that are available are fractionated radiation therapy, which is mostly palliative. And with the successes that CAR-T cell-based therapies have had in hematologic cancers, which have now spilled over to solid tumor therapies, the investigators wondered and asked in this paper whether or not CAR-T cell-based therapies are efficacious for DIPG and diffused midline gliomas as well. And the first publication, which highlights their pre-clinical work, started with donors who supplied tumor samples. And these samples were screened for available cell-based and antibody-based immune therapy using a 250 antibody panel. And the tumor signal that came up, which was the most prevalent in DIPG samples, or diffuse midline glioma samples, was disyloganglioside, or GD2, which is a tumor marker that's found in neural crest tumors. And luckily, they're developing CAR-T cell, as well as antibody therapies available. And so they went on to look at the efficacy of anti-GD2 CAR-T cells using primary cultures from DIPG tumor samples and compared them to CD19 CAR, which is their vehicle control. And what they show in these in vitro experiments is that anti-GD2 CAR-T cell therapy has a pretty significant tumor-cidal efficacy that's dose-dependent. They go on to validate this in an in vivo model. So these are luciferase-expressing DIPG xenografts in mouse. And they infuse them with either anti-CD19 CAR or anti-GD2 CAR-T cells, and then look at bioluminescence to look at progression of the tumor burden. And what you see here is BLI imaging over time. And what they show is 14 days following infusion of anti-GD2 CAR-T cells, there's a significant decrease in the tumor burden that was seen in the GD2 CAR-T arm in comparison to the CD19 CAR arm. And they also show that there's a significant survival benefit in these animals that is seen in this Kaplan-Meier curve. One thing they have noted is that the mortality that's seen on the GD2 CAR-T-infused animals was mostly actually on-target toxicity. And so tumor edema that happens in a very precarious spot, which is the brainstem or spinal cord, causing obstructive hydrocephalus. And so this is something to consider as they transition to the phase one clinical trial, which they have moved pretty fast into currently. And so this is an ongoing phase one trial to look at autologous GD2 CAR-T cells for children who have diffused midline gliomas. And the primary outcomes that they're looking at is manufacturing feasibility, tolerability, and safety. And then the second objective is clinical efficacy, although they're underpowered for this. I want to just highlight the protocol, which really leans on the preclinical findings. And so all of the patients that were enrolled in this study have an Omeya reservoir that's placed not only for direct installment of the CAR-T cells, but also for CSF diversion and settings of on-target toxicity. So what they have shown us in the early findings of their trial is that they're able to manufacture these cells in time for this very aggressive tumor. They've also shown us that there is CAR-T cell kinetics that will have implications for the frequency of infusion. So it seems like the CAR-T cells persist only for about a month, at least in the circulation. They've shown us that there is really no unanticipated adverse outcome with the infusions. However, they have seen a pretty significant cytokine release syndrome, as well as on-target toxicity, which they have coined as tumor inflammation associated neurotoxicity, which has transient worsening of symptoms or obstructive hydrocephalus, which has required aggressive immune modulation and CSF diversion. All to say that these are patients that are going to be in our units being treated as this trial goes forward. The most interesting part of this is that for 75% of their patients, similar to what they saw in the preclinical findings, there have been a significant decrease in their tumor burden, both radiographically as well as with gain of function. And the average survival so far they've reported is about 20 months, which is doubling the historical survival rate for this cancer. In summary, anti-GD2 CAR-T cells seem to demonstrate anti-tumor efficacy in pediatric DMG preclinical models. And the phase one studies seem to be going fairly well with notable on-target toxicities, including tumor inflammation associated neurotoxicity, which will probably require aggressive immune modulation and neuro-intensive care. And so we will likely see these patients as the trial continues to grow. With that, I thank you and I'll take questions at the end.

red blood cells

immune sensors

TLR9 receptors

angiotensin II

anti-GD2 CAR-T cell therapy