Hi, my name is Diego Cruz. I'm a Pediatric Infectious Disease Fellow at Nationwide Children's Hospital, and I'm very excited about the opportunity to share our work. In the setting of sepsis, the immune response can generate a pro-inflammatory cytokine cascade that produces the classic signs and symptoms that we as clinicians are familiarized with of a fever, capillary leak, and organ dysfunction. However, nearly simultaneously, there is a compensatory anti-inflammatory response than when it's severe, is termed immune paralysis. It is important to note that immune paralysis has been associated with an increased risk of adverse clinical outcomes in children with septic shock, as nosocomial infections, prolonged multi-organ dysfunction, and death. However, the bulk of the work of immune paralysis in children has focused on the innate immune response. We know that whole blood, when stimulated ex vivo with LPS, a potent monocyte stimulant, should produce large quantities of TNF-alpha, a pro-inflammatory cytokine, and we also know that when in the setting of immune paralysis, it is characteristic to have a severe reduction in this TNF-alpha response. That's why in our essay that has been validated in the literature, immune paralysis can be identified when we find a TNF-alpha response less than 200 picograms per ml after a stimulation of whole blood with LPS. However, this is only a representation of the innate immune system, and the adaptive immune response is likely to be important as well, but is much less well understood. For example, in this paper from the group of Dr. Wong, they were able to measure the expression of 100 subclass of defined genes in pediatric patients with septic shock. And what I want to highlight here is that they were able to identify two subclasses or endotypes within the cohort. And the group that you are seeing on the left, that is the subclass A, you can see that in this heat map, they have a higher degree of blue, and this is because there was repression of several of the genes that corresponded to downregulation of the adaptive immune system. And this group with the downregulated adaptive immune response had a higher mortality and a higher complicated curse compared to the group that had a more pro-inflammatory response in the genes of the adaptive immune function. There is also evidence that probably we have hypoactive cells in the setting of septic shock. In this pilot study conducted by our group with 22 children with septic shock that were sampled within 48 hours of shock onset, it was identified that children that had persistency of an infection or that developed nociclonal infections in the setting of septic shock had a lower absolute lymphocyte count, but more importantly, had a lower interferon gamma production capacity after stimulation of lymphocyte TCD4 with PHA, that is a potent lymphocyte stimulant. However, it is worth asking, what about other cytokines different to interferon? This is why we need to better characterize adaptive markers of immune paralysis that could also drive interventions to reverse sepsis-induced immune suppression. And I say this because with our understanding of the innate immune system in the setting of immune paralysis, we have been able to conduct studies that have shown promising results to reverse immune paralysis as the one shown in this graphic with GN-CSF being used. And today, we are using TNF-alpha response to identify septic children with immune paralysis and enroll them in clinical trials of personalized immune modulation. But our current gap in the literature is that we still poorly understand biomarkers of lymphocyte function in the setting of immune paralysis. And this leads to the hypothesis of our study that is that septic children with immune paralysis will have a concurrent suppression of lymphocyte cytokine production capacity. So what I'm going to show you are the results of a prospective observation study of children with septic shock admitted to the pediatric ICU at Nationwide Children's Hospitals. And you can see here our inclusion and our exclusion criteria, but what I want to highlight in this slide is that all patients are consent, enrolled, and sampled within four-year hours of sepsis onset. These samples are then used for two ex vivo stimulation assays. The first one represented in this graphic is one for evaluation of the innate immune function in which we take whole blood, stimulate it with LPS, incubate it, and then measure TNF-alpha. And then we identify children with immune paralysis as those that had a TNF-alpha production capacity less than 200 micrograms per ml. The second assay evaluates that active immune system by a stimulation with PHA, apothen lymphocyte stimulant, and after incubation, we measure all these cytokines. We have been able to enroll two different cohorts, a cohort of children with septic shock and a cohort of healthy outpatient controls. Our first step was to evaluate the TNF-alpha production capacity, and you can see in this graphic in the y-axis, the TNF-alpha response, and then two different groups. In red, the septic group, and in green, the healthy control group. And as you can see, patients with septic shock had a significant decrease in the TNF-alpha response. Then we evaluated among our septic cohort the TNF-alpha production capacity, and we were able to identify those children that had immune paralysis. We were able to identify six children with demonstrated immune paralysis, again, characterized by the TNF-alpha response less than 200 micrograms per ml. What we did next was to analyze our data from the PHA assay to evaluate adaptive immune function. So what you can see in this graphic is that in the y-axis, we have the concentration of the cytokine production capacity, and then in the x-axis, we have different cytokines that we measured that we have grouped by phenotype of lymphocyte production. Then what I want to highlight in this graphic is that you can see that we found several cytokines for which the production capacity was decreased when we compared septic children against those that were healthy controls. And more importantly, what I want to highlight is that there is no more pronounced decrease in either of the profiles, Th1 or Th2, and it seems to represent a more global downregulation of the cytokine production capacity. Lastly, what we did is to evaluate our cytokine production capacity from our PHA assay, classifying or subclassifying our cohort in three different groups, those with immune paralysis represented in red, those without immune paralysis represented in yellow, and those that were healthy controls represented in green. And you can see these cytokines, all these were the ones that had a significant decrease in cytokine production capacity when compared with healthy controls. But more importantly, the two graphics that are in the top represent the cytokine production capacity that were decreased only in children with immune paralysis when compared against healthy control, while the three cytokines that are in the bottom of the graphic were decreased both in the immune paralyzed group and the non-immune paralyzed group when compared against healthy controls. Now, what are the take-home messages? First, immune paralysis in septic children is associated with a suppression of both the innate and the adaptive immune response. Two, the PHA-induced cytokine production capacity seems to be decreased in children with septic shock, but this probably does not simply represent an eschewing towards a Th2 phenotype. Third, the patients with immune paralysis may have a different profile of decreased PHA-induced cytokine production capacity when compared with patients that don't have immune paralysis. And more importantly, we believe that this approach could be used to drive enrollment into future clinical trials of immunomodulatory therapies. And additionally, that these readouts of adaptive immune function may also serve as biomarkers of the recovery of immune function in the setting of such clinical trials. With that, I want to finish my presentation acknowledging the intramural funding program at Nationwide Children's Hospital, all the whole lab team members and critical care research coordinators for their invaluable work, all the patients and families that make this study possible, and lastly, the SCCM committee for selecting our work and giving us the opportunity to speak to you today. Thank you very much.

immune paralysis

septic shock

innate immune response

adaptive immune response

biomarkers of lymphocyte function