Hello, and thanks for joining today, and thank you to SSCM for having me speak today on a topic that's near and dear to my heart, specifically, we'll be talking about COVID-19 disease and immune response. I'm Ken Remy, I'm an Associate Professor in Internal Medicine, Pediatrics, Biochemistry and Pathology in the divisions of Pulmonary Critical Care Medicine and Pediatric Critical Care at Rainbow Babies and Children's University Hospitals of Cleveland and Case Western. And yes, I do have a palm tree in the background just to simulate if we were actually able to have gone to Puerto Rico, I hope to see many of you as we enjoy the warmth of your company in the future. But we're going to talk about the immune response. And sadly, this is a topic that likely could go on for days with a lecture that I could probably work with a different people to probably talk about different aspects of both innate and adaptive, DCs and NK cells and T cells and a number of other soluble modifiers of the disease. So I'm going to try to summarize at least some of the data that we have from our laboratory, some of the review of some of the key modifiers, and then we'll end with what I think would be an important consideration. So in deference right now for this talk, I want to just make sure that COVID-19 everyone was and as I refer to SARS-CoV-2, we'll be talking about that as the virus today. So as everyone here knows, we're quite familiar with the structure of the coronavirus, which is made up of spike protein, envelope protein, nucleocapsid proteins, membrane glycoprotein and certainly the ability for MRNA and subsequent transcription. As everyone here also knows that it's many of these areas that specifically, and especially the nucleocapsid protein that are actively engaged and involved in a number of different vaccine technologies that have been helpful in this. This is adapted from the WHO update and as I was asked to talk a little bit about host immune response, I was asked to give some preliminary discussions about some of the background about the immune system. And so I think that these next couple of slides certainly, I hope, will capture that information. So the body's normal immune response to viral infection is to defend against these pathogens and resist them. And there's two types of immunity, there's both innate and adaptive immunity, and they're not actually against one another, they actually have significant interplay. And they also have likely at all given times significant interplay that's just not an isolation that one system works while the other one is on pause waiting for the other one to kick in. In fact, both of them work in conjunction, and are vitally important as we will talk about with COVID-19 disease. The innate immune response is the first line of defense classically thought to be the general immediate responder to any infection, where innate immune cells will secrete interference cytokines and chemokines. They're responsible in mass if you think about macrophages and monocytes and even neutrophils for cytokine production that we would classically say would be part of the cytokine storm. And these interference will interfere with virus replication. The adaptive immune response is our second line of defense where there are specific responses to infection usually starts about six to eight days afterwards, and it's mediated by both T cell for cellular mediated immunity as well as B cell which is antibody responses. The innate immune response is immediate, where the cellular and antibody response usually starts after six to eight days. Here you can see the incubation period and just for general viral infections. And you can see that there's persistent antibody late antibody IgG levels here as delineated in purple. The innate immune response is made predominantly up of macrophages neutrophils and case cells and DCs. When a virus enters the body cells can recognize the markers present on the virus. This was results in a non specific viral activity. And then these cells of the innate immune system are activated to remove these pathogens and foreign cells from the body and activate an adaptive immune response. And the adaptive immune response we've got two types of cells are T cells provide our cellular response where they recognize cells that are infected with a specific virus. They rapidly increase in number to tackle the infection. We have CD4 helper T cells which bring other cells of the immune system together and stimulate B cells to produce antibodies to that virus. And two we've got CD8 cytotoxic T cells that kill the cells in which the virus is multiplying and help to slow down or stop the infection. Now absolutely we're simplifying CD4 and CD8 and I'm not talking about further subclasses or subsets, but for the purposes of this talk I think an understanding of at least these terms is helpful. For B cells the antibody response these cells produce antibodies are specific to the virus IgM antibodies. They're the first to come and disappear after a few weeks with IgG antibodies produced a couple days later with titers usually remaining for months or later. And then we have memory cells that once the infection is over these T cell B cells decline in number but some cells remain these memory cells and they'll rapid response if they come in contact with the same virus again killing the virus and accelerating an antibody response. Let's discuss a little bit about the SARS-CoV-2 virus. So the virus will attempt to enter via usually the nasal passages and then will attempt to bind an endocytosis via TMPRSS2 with other entry factors and ACE2 receptor as well. And this allows for host internalization. This then subsequently then has a number of different pathways that will move towards an endosome. And then subsequently will go through host ribosomal and genomic RNA transcription complex for replication. This genomic RNA will then subsequently move towards the endoplasmic reticulum where it will then induce eventually exocytosis and then induce inflammatory disease modulators namely increases in IL-6, TNF-alpha, CCL-2, 3 and 10 and others. This will begin that cytokine storm if you will, this over activation of the immune system and then subsequently will be the trigger for downstream effects with the adaptive immune system. In regards to internalization, this is a really exquisite paper that came out in Nature Biotechnology in 2021. And this was I think done namely after the original JAMA paper was done in 2020 that demonstrated that younger children had lower quantities of ACE2 receptors as to their older children and adult counterparts. There have been some conflicting studies that have certainly looked at the quantity of ACE2 receptors in children as compared to adults. But this exquisite study certainly looked at a number of different subsets against healthy and sick adults versus healthy and sick children, then did single cell RNA sequencing and then evaluated a number of different cell subtypes. And what was quite interesting in this study is that the authors reported that children do have in fact a fair amount of viral burden in their airway, but their airway immune cells are primed for virus sensing. So they have a very robust early innate antiviral response to SARS-CoV-2 infection in adults, and this helps for early clearance. This is at least data that came before the Delta variant, so it's unclear at this point if this data has been recapitulated with Delta variant or Omicron, but certainly that would be quite interesting and I await that finding. In Nature Medicine in 2021, this review demonstrated what the pathophysiologic factors of COVID-19 are. And so if you think about the host immune response, you've got triggers, you've got mediators and you've got effector cells. And so as we previously just have mentioned, SARS-CoV-2 with ACE2 on the cell receptor will then induce a whole host of anti-spike antibodies. There'll be extracellular to like receptor re-signaling. This goes through PAMP signaling, and then there'll be mediation through neutrophils, subsequently complement and macrophage monocytes, T lymphocytes with generation from macrophages of pro-inflammatory cytokines, which then will induce subsequent downstream lymphopenia. This lymphopenia is vitally important because most, and I'll show you in a second, most patients that are in the intensive care unit, especially with increased severity, this is an excellent marker of the robustness of their macrophage pro-inflammatory overactivation condition that subsequently will lead towards profound exhaustion of T cells and or immune exhaustion. And then subsequently, as this moves towards effector cells, there's endothelial activation alterations in the coagulation hemostatic pathways that lead towards subsequently downstream effects clinically in patients that develop pulmonary fibrosis. And if you were to further look at the hemostasis and coagulation pathway at the level of the alveoli, as SARS-CoV-2 traverses the alveolar interstitial barrier into the endothelial, you'll see a fair amount of complement activation and neutrophil extracellular traps or nets, if you will, that will then involve with the ability for pro-coagulation, which is what leads towards outpatients to have increases in D-dimer, there's thrombus generation, and subsequently there's the endothelial cell dysfunction with a number of different modifiers that will lead towards increased thrombosis, vasoconstriction increases, fibrosis, and then ongoing capillary leak with endothelial dysfunction and increased inflammation. So what is the host immune trajectory? Well, this paper, it came out of the International Archives of Allergy and Immunology in 2021 in response to specifically severe COVID-19 disease. On the y-axis, you'll see severity of COVID-19. On the x-axis, you'll see both the viral to the host inflammatory response. And as severity increases, you traverse an increase into the hyper-inflammatory phase, which subsequently will give you downstream nonspecific biomarker elevations, as well as some specific cytokine, yet descriptive interleukin-6 increases in some patients. So if we take a historic, you know, back-in-time trip to when the initial papers from Wuhan, China, were being developed in JAMA and Lancet and in New England Journal of Medicine, this one really caught my eye because it discussed the 138 hospitalized patients with COVID-19. And what I find most interesting is it said the most common laboratory abnormality it's observed was total lymphocyte depression or lymphopenia, and that these lymphocyte counts continue to decrease until after death occurred. And this, they thought, was associated with cellular immune deficiency. So it certainly is consistent with the possibility that there is some degree of cytokine storm. I hate that term, but it's a nonspecific term that certainly needs more refinement. But an overactivation of the underlying innate immune response with then interplay with the decreasing the adaptive immune response, i.e. T-cell exhaustion, certainly could demonstrate lymphopenia. And what was really interesting about these early findings was that when you compared non-survivors to survivors, those that died had profound lymphopenia. And interestingly, as time elapsed from days after disease onset, these patients had a rise in polymorphic nuclear cell counts, which then led to people believing that they might have secondary infections. And in fact, in JAMA, they reported about one half of those patients within the intensive care unit did, in fact, develop a secondary infection. With lymphopenia the most common lab finding and continuing to decrease until death, this certainly gave at least insight that perhaps a cytokine storm and isolation was not the necessary cause of death, but perhaps the T-cell exhaustion had a predominant effect that subsequently led towards mortality. Two additional groups, both Chen and JCI published early in Jeanette and ICM in 2020, and they looked specifically at the same concept. So absolute lymphocyte count over days in the intensive care unit. You can see that there, as compared to healthy controls, profound immune suppression between T-cell CD3 subsets, CD4 subsets, CD8 cell and B cells and NK cells with absolute lymphocyte counts much lower than their control population. And there certainly was relevant differences between severity of illness with purple here with severity of illness, higher severity of illness and moderate cases being next to that across different cell subtypes. So our group, when the pandemic started, decided that we also wanted to look at our patient population. And so we were fortunate to early in this, in the pandemic to be able to discover some key findings we think that were helpful. The first one is that we compared COVID ICU patients to septic ICU patients to critically ill non-septic patients to healthy volunteers. And as you can see here, most of the groups were pretty evenly matched across the board and we were able to make some key considerations with doing some immune profiling of these patients. But to be able to do that, and our group is made up of Monty Mazur, Isaiah Turnbull and Richard Hotchkiss with collaborations with Chip Caldwell at the University of Cincinnati and Blink Moldauer, Tyler Loftus and Scott Brackenridge at the University of Florida and University of Washington. We were able to make some key determinations that if you look at the survivors versus non-survivors, we recapitulated that earlier Wuhan data. As time elapsed with days after ICU admission, a prolonged lymphopenia was associated with mortality. And interestingly, red dots are non-survivors, black dots are survivors. This is COVID-19 patients over the duration of their intensive care unit stay against septic patients over their duration. And one thing stands out. Undeniably, although there's profound lymphopenia, IL-6 levels were approximately two-fold higher than in septic patients. So next we wanted to understand specifically what was happening with two markers of innate and adaptive immune function. And so our laboratory utilizes enzyme-linked immune absorbent spot or ELI spot. And what you see here is a 96-well plate that you can plate either whole blood or you can isolate PBMCs. You can stimulate these cells with lipopolysaccharide for production of TNF-alpha or stimulate them with CD3, CD28, which would produce interfering gamma. These are surrogates for adaptive and innate immunity, respectively. And you count the number and size of the spots, which tells you the assessment of that cellular response and their function. And so if you have lots of spots after the positive control, that would be consistent with a hyperinflammatory response to that positive control. But if you have very limited response, then that would be consistent with potentially suppression. And you can also expose these cells to different therapies. And so what we found here, and this is just representative healthy versus COVID patients, this is just a representative figure, we looked at interfering gamma production. We compared our COVID-19 patients with red dots being non-survivors, black dots being survivors, compared to our septic patients and our critically ill non-septic. And across the board, you can see there is significantly depressed T cell function on these COVID-19 patients. And this data certainly reflected that those that died had certainly more immune exhaustion or immune suppression associated with interfering gamma production after CD3, CD28 stimulus. We next went on to compare interfering gamma and TNF alpha production in those same cohorts. And once again, TNF alpha production was drastically and dramatically decreased as compared to its septic cohorts, critically ill non-septic, and our healthy volunteers. Which really was kind of interesting because the world is talking about a cytokine storm. And although we did see one isolated cytokine that was elevated in interleukin 6, we were seeing a predominant phenotype across the board, especially those that are dying, that they have immune paralysis. And we followed this over time. And you just have one dot here that brings this up. But over time, interfering gamma production and TNF alpha production in our cohort was certainly reduced in comparison to those that died compared to those that lived. Ali Elabadi and Phil Mudd, also collaborators with the same patient population and some additional patients, compared this SARS-CoV-2 suppression that we saw, that they also recapitulated and compared it against their work in influenza. And they were able in different CD8 and CD4, both activated and unactivated or unstimulated T-cells and B-cells with classic and intermediate non-classical monocytes. They saw a profound immune suppression across all cell types. Isaiah Turnbull has taken this even one step further and wanted to understand specifically proteomic signatures and using CyTOF evaluated severe versus moderate COVID-19 patients, as well as also did some key luminex cytokines and ELI spot data to confirm some of the previous work we had. And Isaiah demonstrated what I think is really interesting. He showed not only did COVID induce a systemic inflammatory response, as you can see across severity, red is severe, blue is healthy donor, and orange is your moderate. And you can see in our group, there was an increase in severity associated with interleukin 6. So perhaps if you take the most severe, you'll see the most over-exaggerated innate response. Whereas if they're less sick, they perhaps may have less of a response as in comparison to healthy donors. But this was a phenomenon seen across the board. 7 out of 36 of those cytokines were significantly elevated in severe COVID-19. And next, Dr. Turnbull evaluated a number of different signaling phosphoproteomes, including STAT pathways, NF-kappa B pathways, and looked at neutrophils, monocytes, CD4, CD8, and NK cells. And this is the big thing that came out of his work that's currently accepted for publication in PLOS One. He found that STAT3 is the predominant signaling pathway activated by COVID-19. Why is this important? Well, STAT3 sits at the intersection of inflammation of the innate antiviral immunity. So it inhibits type 1 antiviral responses. And these type 1 interference signatures are dramatically repressed in peripheral blood leukocytes of critically ill patients with COVID-19. Furthermore, STAT3 knockout mice have defects in bacterial clearance during bacterial sepsis, but an exaggerated systemic inflammatory response. Sound familiar? So pharmacologic inhibition of STAT3 attenuates that inflammatory cytokine production during sepsis and is associated with improved survival. Dr. Turnbull went on to further correlate this negatively, STAT3 phosphorylation with T-cell interfering gamma production. But this is what's really interesting based off of Dr. Turnbull's findings. Varsitinib, a therapy that we currently use quite frequently in these patients, inhibits STAT3 and attenuates the inflammatory cytokine production during sepsis with improved survival. Starting to make sense now that we've got phosphoproteomic data for STAT3 and we've got a therapy that physiologically now makes sense why it would actually improve outcomes in patients. John Weary's group at UPenn further developed three COVID immune phenotypes. We've talked a lot about the host response with some patients having robust cytokine storm with interleukin 6 elevations and some other subsets. We've also demonstrated that there's a pretty profound immune suppression or T-cell exhaustion. And so you've got this phenomenon where you've got over-activation and hypo-inflammation. And so depending on which type of patient and where they are specifically in their disease, temporal evolution, they may in fact have very different immune phenotypes. So John's group specifically characterized these groups with deep immune profiling techniques and demonstrated that one immune phenotype had highly activated CD4 and CD8s, activated CD8 EMRs and PV responses. This would be an over-activation group. He demonstrated also that one group had very low or no activated T-cells or B-cells, which would demonstrate a very suppressed or paralyzed group and an intermediate group. But this is the problem. How do we know where a patient is? This looks very similar to sepsis. How can we know which patient will have which response and then tailor our therapies to that individual person? Because this would be a game changer with precision medicine, because we do know that not every therapy works in every patient. If you treat it all as a one-size-fits-all, you're invariably going to have mixed results on your studies, especially when we know that COVID-19, for instance, induces two clinical phenotypes that are very different with hypoxemia. You've got a high respiratory compliance group and you've got a low respiratory compliance group. And they're very different, and they're treated potentially differently in the intensive care unit. But understanding these clinical phenotypes against specific immune phenotypes may be really helpful because you may alter your immune modulating therapies. So one way that our group, as mentioned with ELISPOT, has tackled this problem is by using ELISPOT to model COVID-19 disease. After stimulation with CD3 and LPS, we can evaluate interferon gamma and TNF alpha, and we can look at what the spot size is for each of these and whether or not there's down or up expression of those cytokines. And we can take blood from the patient. We can evaluate the response. For instance, if it's low, as you see here, we can then perhaps give them the positive stimulation, figure out in the dish which therapies actually could influence the response of their hypo-inflamed. You give them a specific therapy in this dish. You can see the response in real time. And then you can then go back to the patient and give them that therapy. And that's really exciting. And so we evaluated a number of patients across different therapies. So what you're looking at here is actual ELISPOT wells of a patient. This is CD3, CD28. This is the, we run in duplicate. This is IL-7, interferon beta, interferon alpha, ox40, and a checkpoint inhibitor. And you can see here that from the 20 and 35 cells, there's not much of response to these therapies. When we looked at a different patient after their positive production with CD3, CD28 for interfering gamma production specifically here, you can note that the patient had about 260 cells, spot sizes, if you will. And these other therapies really didn't have a meaningful response. In fact, some of them knocked down that cell. In fact, were anti-inflammatory. But with interleukin-7, you can see that we dramatically increased the number of spot cells of interferon gamma production. And that patient's ex vivo sample. And so we did this. We were able to give this experimental drug to this gentleman and he lived. This precision medicine approach to really being able to define which patients at a single time point or multiple time points perhaps might benefit from specific therapies directed against both adaptive and innate markers may in fact be helpful. We're going to switch gears here for a second. And this is adapted from James Rothman's exceptional presentation. And here he looked at host response antibody development, which is really short-lived immunity. And you're looking at here the IgG levels of the acute phase and convalescent phase and a number of patients is published in Long and Nature Medicine in 2020. And you're looking at the neutralization rate. And what's interesting is about one in five patients recover from SARS-CoV-2 infection without any detectable antibody titer, highlighting the importance of cell-mediated immunity. And what's most interesting is that antiviral T cell responses may even be more critical. I.e., we may be using anti-inflammatory therapies to suppress patients as they're inflamed. But it's certainly feasible that those therapies for some patients may in fact cause perturbations in the cell-mediated immune response for these patients and make them further hypo-inflammed. And furthermore, in cell in 2020, it was interesting because in these same patients, these patients had long-lasting and robust CD4 and CD8 T cell responses, but they didn't have antibody response. So clearly host immune response in these patients is challenging. You've got different innate and adaptive immune responses in some patients. Invariably, there's interplay between both systems. You've got activation in some patients, suppression in others. You've got alterations in the immune status, changes in epigenetics, changes in checkpoints and Tregs. You've got changes in your metabolism and your microenvironment. And so it can be challenging certainly to decipher where we are with a person's acute response in COVID and how that perhaps may influence their downstream effects with long COVID. Undeniably, SARS-CoV-2 infection is complicated. It provides a heterogeneous set of host immune responses that can be further characterized into immune and clinical endotypes to inform therapy. I think it's relevant and important to understand both pro and hypo-inflammatory conditions, which will be paramount for effective therapy administration. Antiviral T cell responses are likely critical for recovery, and we should understand how therapies may influence long-term immune consequences, including in long COVID and secondary infections to assess acute versus long-term efficacy. Specific immune biomarkers should be evaluated for both clinical care and future research therapeutic development. Certainly, as we move on from this pandemic, precision medicine certainly should carry the day, in my view, so we can move away from this one size fits all. And so functional readouts for hemostasis and other areas are going to be relevant as we move forward. And with that, I say thank you. And I hate to tease you with this palm tree behind me, but I look forward to any questions at the end of this session. And thank you very much for joining me, and thank you to the Society of Critical Care Medicine for having me give this talk today.

immune response

COVID-19

disease progression

innate immunity

adaptive immunity

viral infections