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Purpose: To study the biology and identify markers of severe cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS) in children after chimeric antigen receptor T-cell (CAR T) treatment. Experimental Design: We used comprehensive proteomic profiling to measure over 1,400 serum proteins at multiple serial timepoints in a cohort of patients with B-cell acute lymphoblastic leukemia treated with the CD19-targeted CAR T CTL019 on two clinical trials. Results: We identified fms-like tyrosine kinase 3 (FLT3) and mast cell immunoglobulin-like receptor 1 (MILR1) as preinfusion predictive biomarkers of severe CRS. We demonstrated that CRS is an IFNγ-driven process with a protein signature overlapping with hemophagocytic lymphohistiocytosis (HLH). We identified IL18 as a potentially targetable cytokine associated with the development of ICANS. Conclusions: We identified preinfusion biomarkers that can be used to predict severe CRS with a sensitivity, specificity, and accuracy superior to the current gold standard of disease burden. We demonstrated the fundamental role of the IFNγ pathway in driving CRS, suggesting CRS and carHLH are overlapping rather than distinct phenomena, an observation with important treatment implications. We identified IL18 as a possible targetable cytokine in ICANS, providing rationale for IL18 blocking therapies to be translated into clinical trials in ICANS.

PDF file - 713K, Figure S1. Kinetics of EL4 killing. Figure S2. iNKT cells kill several CD1d-expressing targets. Figure S3. EL4s express low levels of NKG2D ligands while iNKT and NK cells express NKG2D. Figure S4. Thymic but not splenic NKT cells kill EL4s in a GAg-dependent manner. Figure S5. DN3A4-1.2 NKT hybridoma cells kill GAg-loaded tumors. Figure S6. CD4+ and CD4- 8- liver iNKT cells possess cytotoxic capacity. Figure S7. iNKT cell reconstitution of NSG mice is not associated with in vivo accumulation of contaminating NK cells.

Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we found that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially induced by iNKT cell agonists of varying T-cell receptor (TCR) affinities, such as OCH, α-galactosyl ceramide, and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of TCR signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell–deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T lymphoma. Cancer Immunol Res; 2(1); 59–69. ©2013 AACR.

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Supplementary Figure from Comprehensive Serum Proteome Profiling of Cytokine Release Syndrome and Immune Effector Cell–Associated Neurotoxicity Syndrome Patients with B-Cell ALL Receiving CAR T19

Purpose:To study the biology and identify markers of severe cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS) in children after chimeric antigen receptor T-cell (CAR T) treatment.Experimental Design:We used comprehensive proteomic profiling to measure over 1,400 serum proteins at multiple serial timepoints in a cohort of patients with B-cell acute lymphoblastic leukemia treated with the CD19-targeted CAR T CTL019 on two clinical trials.Results:We identified fms-like tyrosine kinase 3 (FLT3) and mast cell immunoglobulin-like receptor 1 (MILR1) as preinfusion predictive biomarkers of severe CRS. We demonstrated that CRS is an IFNγ-driven process with a protein signature overlapping with hemophagocytic lymphohistiocytosis (HLH). We identified IL18 as a potentially targetable cytokine associated with the development of ICANS.Conclusions:We identified preinfusion biomarkers that can be used to predict severe CRS with a sensitivity, specificity, and accuracy superior to the current gold standard of disease burden. We demonstrated the fundamental role of the IFNγ pathway in driving CRS, suggesting CRS and carHLH are overlapping rather than distinct phenomena, an observation with important treatment implications. We identified IL18 as a possible targetable cytokine in ICANS, providing rationale for IL18 blocking therapies to be translated into clinical trials in ICANS.

Supplementary Data from Comprehensive Serum Proteome Profiling of Cytokine Release Syndrome and Immune Effector Cell–Associated Neurotoxicity Syndrome Patients with B-Cell ALL Receiving CAR T19

People with spinal cord injury (SCI) get recurrent infections, like urinary tract infections and pneumonias, that cause mortality and worsen neurological recovery. Over the past decades, researchers have proposed that post-SCI lymphopenia and decreased lymphocyte function increase susceptibility to infections and worsen neurological outcome in humans, leading to a condition called SCI-Induced Immune Depression Syndrome. In this review, we explore how SCI affects blood lymphocyte homeostasis and function in humans and rodents. Understanding how SCI affects blood lymphocytes will help the management of recurrent infections in spinal cord injured people and shed light on the clinical translation of findings in animal models to humans.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of severe immune dysregulation that encompasses a broad range of underlying genetic diseases and infectious triggers. Monogenic conditions, autoimmune diseases, and infections can all drive the phenotype of HLH and associated immune hyperactivation with hypercytokinemia. A diagnosis of HLH usually requires a combination of clinical and laboratory findings; there is no single sensitive and specific diagnostic test, which often leads to "diagnostic dilemmas" and delays in treatment initiation. Ferritin levels, one of the most commonly used screening tests, were collected across a large tertiary care pediatric hospital to identify the positive predictive value for HLH. Herein, we present several cases that illustrate the clinical challenges of confirming an HLH diagnosis. Additionally, we report on the utility of establishing a formal multi-disciplinary group to aid the prompt diagnosis and treatment of patients presenting with HLH-like pathophysiologies.

The ability to utilize preclinical models to predict the clinical toxicity of chimeric antigen receptor (CAR) T cells in solid tumors is tenuous, thereby necessitating the development and evaluation of gated systems. Here we found that murine GD2 CAR-T cells, specific for the tumor-associated antigen GD2, induce fatal neurotoxicity in a costimulatory domain-dependent manner. Meanwhile, human B7H3 CAR-T cells exhibit efficacy in preclinical models of neuroblastoma. Seeking a better CAR, we generated a SynNotch gated CAR-T, GD2-B7H3, recognizing GD2 as the gate and B7H3 as the target. GD2-B7H3 CAR-T cells control the growth of neuroblastoma in vitro and in metastatic xenograft mouse models, with high specificity and efficacy. These improvements come partly from the better metabolic fitness of GD2-B7H3 CAR-T cells, as evidenced by their naïve T-like post-cytotoxicity oxidative metabolism and lower exhaustion profile., Antibodies targeting a tumor antigen, GD2, show some efficacy for neuroblastoma but induce severe neuropathic pain and peripheral neuropathy. Here the authors design a gated chimeric antigen receptor (CAR), using GD2 as the gate and another tumor antigen, B7H3, as the target, to find this GD2-B7H3 CAR capable of suppressing neuroblastoma in mouse models with little adverse effects.

Key Points sC5b9 plasma levels are elevated in children with SARS-CoV-2 infection, even if they have minimal symptoms of COVID-19. A high proportion of children with SARS-CoV-2 infection met clinical criteria for TMA., Most children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have mild or minimal disease, with a small proportion developing severe disease or multisystem inflammatory syndrome in children (MIS-C). Complement-mediated thrombotic microangiopathy (TMA) has been associated with SARS-CoV-2 infection in adults but has not been studied in the pediatric population. We hypothesized that complement activation plays an important role in SARS-CoV-2 infection in children and sought to understand if TMA was present in these patients. We enrolled 50 hospitalized pediatric patients with acute SARS-CoV-2 infection (n = 21, minimal coronavirus disease 2019 [COVID-19]; n = 11, severe COVID-19) or MIS-C (n = 18). As a biomarker of complement activation and TMA, soluble C5b9 (sC5b9, normal 247 ng/mL) was measured in plasma, and elevations were found in patients with minimal disease (median, 392 ng/mL; interquartile range [IQR], 244-622 ng/mL), severe disease (median, 646 ng/mL; IQR, 203-728 ng/mL), and MIS-C (median, 630 ng/mL; IQR, 359-932 ng/mL) compared with 26 healthy control subjects (median, 57 ng/mL; IQR, 9-163 ng/mL; P < .001). Higher sC5b9 levels were associated with higher serum creatinine (P = .01) but not age. Of the 19 patients for whom complete clinical criteria were available, 17 (89%) met criteria for TMA. A high proportion of tested children with SARS-CoV-2 infection had evidence of complement activation and met clinical and diagnostic criteria for TMA. Future studies are needed to determine if hospitalized children with SARS-CoV-2 should be screened for TMA, if TMA-directed management is helpful, and if there are any short- or long-term clinical consequences of complement activation and endothelial damage in children with COVID-19 or MIS-C., Visual Abstract

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibody responses in children remain poorly characterized. Here, we show that pediatric patients with multisystem inflammatory syndrome in children (MIS-C) possess higher SARS-CoV-2 spike immunoglobulin G (IgG) titers compared with those with severe coronavirus disease 2019, likely reflecting a longer time since the onset of infection in MIS-C patients.

Chimeric antigen receptor (CAR) T-cells directed against CD19 have drastically altered outcomes for children with relapsed and refractory acute lymphoblastic leukemia (r/r ALL). Pediatric patients with r/r ALL treated with CAR-T are at increased risk of both cytokine release syndrome (CRS) and sepsis. We sought to investigate the biologic differences between CRS and sepsis and to develop predictive models which could accurately differentiate CRS from sepsis at the time of critical illness. We identified 23 different cytokines that were significantly different between patients with sepsis and CRS. Using elastic net prediction modeling and tree classification, we identified cytokines that were able to classify subjects as having CRS or sepsis accurately. A markedly elevated interferon γ (IFNγ) or a mildly elevated IFNγ in combination with a low IL1β were associated with CRS. A normal to mildly elevated IFNγ in combination with an elevated IL1β was associated with sepsis. This combination of IFNγ and IL1β was able to categorize subjects as having CRS or sepsis with 97% accuracy. As CAR-T therapies become more common, these data provide important novel information to better manage potential associated toxicities.

OBJECTIVE: To provide guidance on the management of multisystem inflammatory syndrome in children (MIS-C), a condition characterized by fever, inflammation, and multiorgan dysfunction that manifests late in the course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and to provide recommendations for children with hyperinflammation during coronavirus disease 2019 (COVID-19), the acute, infectious phase of SARS-CoV-2 infection. METHODS: A multidisciplinary task force was convened by the American College of Rheumatology (ACR) to provide guidance on the management of MIS-C associated with SARS-CoV-2 and hyperinflammation in COVID-19. The task force was composed of 9 pediatric rheumatologists, 2 adult rheumatologists, 2 pediatric cardiologists, 2 pediatric infectious disease specialists, and 1 pediatric critical care physician. Preliminary statements addressing clinical questions related to MIS-C and hyperinflammation in COVID-19 were developed based on evidence reports. Consensus was built through a modified Delphi process that involved 2 rounds of anonymous voting and 2 webinars. A 9-point scale was used to determine the appropriateness of each statement (median scores of 1-3 for inappropriate, 4-6 for uncertain, and 7-9 for appropriate), and consensus was rated as low, moderate, or high based on dispersion of the votes along the numeric scale. Approved guidance statements were those that were classified as appropriate with moderate or high levels of consensus, as prespecified prior to voting. RESULTS: The ACR task force approved a total of 128 guidance statements addressing the management of MIS-C and hyperinflammation in pediatric COVID-19. These statements were refined into 40 final clinical guidance statements, accompanied by a flow diagram depicting the diagnostic pathway for MIS-C. CONCLUSION: Our understanding of SARS-CoV-2-related syndromes in the pediatric population continues to evolve. The guidance provided in this "living document" reflects currently available evidence, coupled with expert opinion, and will be revised as further evidence becomes available.

Background Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition of immune dysregulation. Children often suffer from primary genetic forms of HLH, which can be triggered by infection. Others suffer from secondary HLH as a complication of infection, malignancy, or rheumatologic disease. Identifying the exact cause of HLH is crucial, as definitive treatment for primary disease is hematopoietic stem cell transplant. Adenoviruses have been associated with HLH but molecular epidemiology data are lacking. Methods We describe the clinical and virologic characteristics of 5 children admitted with adenovirus infection during 2018–2019 who developed HLH or HLH-like illness. Detailed virologic studies, including virus isolation and comprehensive molecular typing were performed. Results All patients recovered; clinical management varied but included immunomodulating and antiviral therapies. A genetic predisposition for HLH was not identified in any patient. Adenovirus isolates were recovered from 4/5 cases; all were identified as genomic variant 7d. Adenovirus type 7 DNA was detected in the fifth case. Phylogenetic analysis of genome sequences identified 2 clusters—1 related to strains implicated in 2016–2017 outbreaks in Pennsylvania and New Jersey, the other related to a 2009 Chinese strain. Conclusions It can be challenging to determine whether HLH is the result of an infectious pathogen alone or genetic predisposition triggered by an infection. We describe 5 children from the same center presenting with an HLH-like illness after onset of adenovirus type 7 infection. None of the patients were found to have a genetic predisposition to HLH. These findings suggest that adenovirus 7 infection alone can result in HLH.

BackgroundImmune-mediated lung injury and systemic hyperinflammation are characteristic of severe and critical coronavirus disease 2019 (COVID-19) in adults. Although the majority of severe acute respiratory syndrome coronavirus 2 infections in pediatric populations result in minimal or mild COVID-19 in the acute phase of infection, a small subset of children develop severe and even critical disease in this phase with concomitant inflammation that may benefit from immunomodulation. Therefore, guidance is needed regarding immunomodulatory therapies in the setting of acute pediatric COVID-19. This document does not provide guidance regarding the recently emergent multisystem inflammatory syndrome in children (MIS-C).MethodsA multidisciplinary panel of pediatric subspecialty physicians and pharmacists with expertise in infectious diseases, rheumatology, hematology/oncology, and critical care medicine was convened. Guidance statements were developed based on best available evidence and expert opinion.ResultsThe panel devised a framework for considering the use of immunomodulatory therapy based on an assessment of clinical disease severity and degree of multiorgan involvement combined with evidence of hyperinflammation. Additionally, the known rationale for consideration of each immunomodulatory approach and the associated risks and benefits was summarized.ConclusionsImmunomodulatory therapy is not recommended for the majority of pediatric patients, who typically develop mild or moderate COVID-19. For children with severe or critical illness, the use of immunomodulatory agents may be beneficial. The risks and benefits of such therapies are variable and should be evaluated on a case-by-case basis with input from appropriate specialty services. When available, the panel strongly favors immunomodulatory agent use within the context of clinical trials. The framework presented herein offers an approach to decision-making regarding immunomodulatory therapy for severe or critical pediatric COVID-19 and is informed by currently available data, while awaiting results of placebo-controlled randomized clinical trials.

We present a series of 6 critically ill children with multisystem inflammatory syndrome in children. Key findings of this syndrome include fever, diarrhea, shock, and variable presence of rash, conjunctivitis, extremity edema, and mucous membrane changes.

To provide guidance on the management of Multisystem Inflammatory Syndrome in Children (MIS-C), a condition characterized by fever, inflammation, and multiorgan dysfunction that manifests late in the course of SARS-CoV-2 infection. Recommendations are also provided for children with hyperinflammation during COVID-19, the acute, infectious phase of SARS-CoV-2 infection.The Task Force is composed of 9 pediatric rheumatologists and 2 adult rheumatologists, 2 pediatric cardiologists, 2 pediatric infectious disease specialists, and 1 pediatric critical care physician. Preliminary statements addressing clinical questions related to MIS-C and hyperinflammation in COVID-19 were developed based on evidence reports. Consensus was built through a modified Delphi process that involved anonymous voting and webinar discussion. A 9-point scale was used to determine the appropriateness of each statement (median scores of 1-3 for inappropriate, 4-6 for uncertain, and 7-9 for appropriate). Consensus was rated as low, moderate, or high based on dispersion of the votes. Approved guidance statements were those that were classified as appropriate with moderate or high levels of consensus, which were prespecified before voting.The guidance was approved in June 2020 and updated in November 2020 and October 2021, and consists of 41 final guidance statements accompanied by flow diagrams depicting the diagnostic pathway for MIS-C and recommendations for initial immunomodulatory treatment of MIS-C.Our understanding of SARS-CoV-2-related syndromes in the pediatric population continues to evolve. This guidance document reflects currently available evidence coupled with expert opinion, and will be revised as further evidence becomes available.

Background Cardiac dysfunction is a prominent feature of multisystem inflammatory syndrome in children (MIS‐C), yet the etiology is poorly understood. We determined whether dysfunction is global or regional, and whether it is associated with the cytokine milieu, microangiopathy, or severity of shock. Methods and Results We analyzed echocardiographic parameters of myocardial deformation and compared global and segmental left ventricular strain between 43 cases with MIS‐C ≤18 years old and 40 controls. Primary outcomes included left ventricular global longitudinal strain, right ventricular free wall strain), and left atrial strain. We evaluated relationships between strain and profiles of 10 proinflammatory cytokines, microangiopathic features (soluble C5b9), and vasoactive‐inotropic requirements. Compared with controls, cases with MIS‐C had significant impairments in all parameters of systolic and diastolic function. 65% of cases with MIS‐C had abnormal left ventricular function ( | global longitudinal strain | Conclusions Myocardial function is globally decreased in MIS‐C and not explained by acute stress cardiomyopathy. Cardiac dysfunction may be driven by the relative skew of the immune response toward interleukin‐6 and interleukin‐8 pathways, more so than degree of hyperinflammation, refining the current paradigm of myocardial involvement in MIS‐C.

BackgroundNeuroblastoma is a commonly lethal solid tumor of childhood and intensive chemoradiotherapy treatment cures ~50% of children with high-risk disease. The addition of immunotherapy using dinutuximab, a monoclonal antibody directed against the GD2 disialoganglioside expressed on neuroblasts, improves survival when incorporated into front-line therapy and shows robust activity in regressing relapsed disease when combined with chemotherapy. Still, many children succumb to neuroblastoma despite receiving dinutuximab-based immunotherapy, and efforts to counteract the immune suppressive signals responsible are warranted. Animal models of human cancers provide useful platforms to study immunotherapies. TH-MYCN transgenic mice are immunocompetent and develop neuroblastomas at autochthonous sites due to enforced MYCN expression in developing neural crest tissues. However, GD2-directed immunotherapy in this model has been underutilized due to the prevailing notion that TH-MYCN neuroblasts express insufficient GD2 to be targeted.MethodsTH-MYCN mice were treated with 14G2a (anti-GD2 antibody), isotype antibody, or phosphate buffered saline from day 14 of life until day 100 or signs of morbidity. Survival was recorded, and tumors were isolated in terminal surgeries for analysis of GD2 expression and immune cell frequencies. Tumors from untreated mice were explanted for generation into cell lines, and GD2 expression was recorded with serial passage in tissue culture. Immunocytology and immunoblotting were performed to evaluate for adrenergic and mesenchymal markers of neuroblasts. Survival curves compared using Kaplan-Meier method with a log-rank test for significance. Unpaired two-tailed Student’s t-tests used for comparison of groups in flow cytometry analysis.Results14G2a markedly extends survival in such TH-MYCN mice. Additionally, neuroblasts in 14G2a-treated mice have reduced GD2 expression and fewer macrophage and myeloid-derived suppressor cells in their tumor microenvironments. Neuroblasts in TH-MYCN-driven tumors express GD2 at levels comparable to human neuroblastomas but rapidly lose GD2 expression when explanted ex vivo to establish tumor cell lines. The loss of GD2 expression ex vivo is associated with a transition from an adrenergic to mesenchymal state that is maintained when reimplanted in vivo.ConclusionsOur findings support the utility of the TH-MYCN model to inform GD2-directed immunotherapy approaches for neuroblastoma as well as opportunities to investigate drivers of adrenergic to mesenchymal fate decisions.

Invariant natural killer T (iNKT) cells comprise a unique subset of lymphocytes that are primed for activation and possess innate NK-like functional features. Currently, iNKT cell-based immunotherapies remain in early clinical stages, and little is known about the ability of these cells to survive and retain effector functions within the solid tumor microenvironment (TME) long-term. In conventional T cells (TCONV), cellular metabolism is linked to effector functions and their ability to adapt to the nutrient-poor TME. In contrast, the bioenergetic requirements of iNKT cells – particularly those of human iNKT cells – at baseline and upon stimulation are not well understood; neither is how these requirements affect cytokine production or anti-tumor effector functions. We find that unlike TCONV, human iNKT cells are not dependent upon glucose or glutamine for cytokine production and cytotoxicity upon stimulation with anti-CD3 and anti-CD28. Additionally, transcriptional profiling revealed that stimulated human iNKT cells are less glycolytic than TCONV and display higher expression of fatty acid oxidation (FAO) and adenosine monophosphate-activated protein kinase (AMPK) pathway genes. Furthermore, stimulated iNKT cells displayed higher mitochondrial mass and membrane potential relative to TCONV. Real-time Seahorse metabolic flux analysis revealed that stimulated human iNKT cells utilize fatty acids as substrates for oxidation more than stimulated TCONV. Together, our data suggest that human iNKT cells possess different bioenergetic requirements from TCONV and display a more memory-like metabolic program relative to effector TCONV. Importantly, iNKT cell-based immunotherapeutic strategies could co-opt such unique features of iNKT cells to improve their efficacy and longevity of anti-tumor responses.

Multi-system Inflammatory Syndrome in Children (MIS-C) is a major complication of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic in pediatric patients. Weeks after an often mild or asymptomatic initial infection with SARS-CoV-2 children may present with a severe shock-like picture and marked inflammation. Children with MIS-C present with varying degrees of cardiovascular and hyperinflammatory symptoms. We performed a comprehensive analysis of the plasma proteome of more than 1400 proteins in children with SARS-CoV-2. We hypothesized that the proteome would reflect heterogeneity in hyperinflammation and vascular injury, and further identify pathogenic mediators of disease. Protein signatures demonstrated overlap between MIS-C, and the inflammatory syndromes macrophage activation syndrome (MAS) and thrombotic microangiopathy (TMA). We demonstrate that PLA2G2A is a key marker of MIS-C that associates with TMA. We found that IFNγ responses are dysregulated in MIS-C patients, and that IFNγ levels delineate clinical heterogeneity.

MIS-C is defined by lymphocyte activation, including elevated plasmablast frequencies and marked activation of CX3CR1+ CD8+ T cells., Pediatric COVID-19 following SARS-CoV-2 infection is associated with fewer hospitalizations and often milder disease than in adults. A subset of children, however, present with Multisystem Inflammatory Syndrome in Children (MIS-C) that can lead to vascular complications and shock, but rarely death. The immune features of MIS-C compared to pediatric COVID-19 or adult disease remain poorly understood. We analyzed peripheral blood immune responses in hospitalized SARS-CoV-2 infected pediatric patients (pediatric COVID-19) and patients with MIS-C. MIS-C patients had patterns of T cell-biased lymphopenia and T cell activation similar to severely ill adults, and all patients with MIS-C had SARS-CoV-2 spike-specific antibodies at admission. A distinct feature of MIS-C patients was robust activation of vascular patrolling CX3CR1+ CD8+ T cells that correlated with the use of vasoactive medication. Finally, whereas pediatric COVID-19 patients with acute respiratory distress syndrome (ARDS) had sustained immune activation, MIS-C patients displayed clinical improvement over time, concomitant with decreasing immune activation. Thus, non-MIS-C versus MIS-C SARS-CoV-2 associated illnesses are characterized by divergent immune signatures that are temporally distinct from one another and implicate CD8+ T cells in the clinical presentation and trajectory of MIS-C.

Objective The aim of the study was to compare presenting clinical and laboratory features among children meeting the surveillance definition for multisystem inflammatory syndrome in children (MIS-C) across a range of illness severities. Methods This is a retrospective single-center study of patients younger than 21 years presenting between March 1 and May 15, 2020. Included patients met the Centers for Disease Control and Prevention criteria for MIS-C (inflammation, fever, involvement of 2 organ systems, lack of alternative diagnoses). We defined 3 subgroups by clinical outcomes: (1) critical illness requiring intensive care interventions; (2) patients meeting Kawasaki disease (KD) criteria but not requiring critical care; and (3) mild illness not meeting either criteria. A comparator cohort included patients with KD at our institution during the same time frame in 2019. Results Thirty-three patients were included (5, critical; 8, 2020 KD; 20, mild). The median age for the critical group was 10.9 years (2.7 for 2020 KD; 6.0 for mild, P = 0.033). The critical group had lower median absolute lymphocyte count (850 vs 3005 vs 2940/uL, P = 0.005), platelets (150 vs 361 vs 252 k/uL, P = 0.005), and sodium (129 vs 136 vs 136 mmol/L, P = 0.002), and higher creatinine (0.7 vs 0.2 vs 0.3 mg/dL, P = 0.002). In the critical group, 60% required vasoactive medications, and 40% required mechanical ventilation. Clinical and laboratories features were similar between the 2020 and 2019 KD groups. Conclusions We describe 3 groups with inflammatory syndromes during the SARS-CoV-2 pandemic. The initial profile of lymphopenia, thrombocytopenia, hyponatremia, and abnormal creatinine may help distinguish critically ill MIS-C patients from classic/atypical KD or more benign acute inflammation.

BACKGROUND: Initial reports from the severe acute respiratory coronavirus 2 (SARS–CoV-2) pandemic described children as being less susceptible to coronavirus disease 2019 (COVID-19) than adults. Subsequently, a severe and novel pediatric disorder termed multisystem inflammatory syndrome in children (MIS-C) emerged. We report on unique hematologic and immunologic parameters that distinguish between COVID-19 and MIS-C and provide insight into pathophysiology. METHODS: We prospectively enrolled hospitalized patients with evidence of SARS–CoV-2 infection and classified them as having MIS-C or COVID-19. Patients with COVID-19 were classified as having either minimal or severe disease. Cytokine profiles, viral cycle thresholds (Cts), blood smears, and soluble C5b-9 values were analyzed with clinical data. RESULTS: Twenty patients were enrolled (9 severe COVID-19, 5 minimal COVID-19, and 6 MIS-C). Five cytokines (IFN-γ, IL-10, IL-6, IL-8, and TNF-α) contributed to the analysis. TNF-α and IL-10 discriminated between patients with MIS-C and severe COVID-19. The presence of burr cells on blood smears, as well as Cts, differentiated between patients with severe COVID-19 and those with MIS-C. CONCLUSION: Pediatric patients with SARS–CoV-2 are at risk for critical illness with severe COVID-19 and MIS-C. Cytokine profiling and examination of peripheral blood smears may distinguish between patients with MIS-C and those with severe COVID-19. FUNDING: Financial support for this project was provided by CHOP Frontiers Program Immune Dysregulation Team; National Institute of Allergy and Infectious Diseases; National Cancer Institute; the Leukemia and Lymphoma Society; Cookies for Kids Cancer; Alex’s Lemonade Stand Foundation for Childhood Cancer; Children’s Oncology Group; Stand UP 2 Cancer; Team Connor; the Kate Amato Foundations; Burroughs Wellcome Fund CAMS; the Clinical Immunology Society; the American Academy of Allergy, Asthma, and Immunology; and the Institute for Translational Medicine and Therapeutics.

Neuroblastoma (NB) is the most common extracranial solid tumor in children and, in the high-risk group, has a 5-year mortality rate of ~50%. The high mortality rate and significant treatment-related morbidities associated with current standard of care therapies belie the critical need for more tolerable and effective treatments for this disease. While the monoclonal antibody dinutuximab has demonstrated the potential for immunotherapy to improve overall NB outcomes, the 5-year overall survival of high-risk patients has not yet substantially changed. The frequency and type of invariant natural killer T cells (iNKTs) and natural killer cells (NKs) has been associated with improved outcomes in several solid and liquid malignancies, including NB. Indeed, iNKTs and NKs inhibit tumor associated macrophages (TAMs) and myeloid derived suppressor cells (MDSCs), kill cancer stem cells (CSCs) and neuroblasts, and robustly secrete cytokines to recruit additional immune effectors. These capabilities, and promising pre-clinical and early clinical data suggest that iNKT- and NK-based therapies may hold promise as both stand-alone and combination treatments for NB. In this review we will summarize the biologic features of iNKTs and NKs that confer advantages for NB immunotherapy, discuss the barriers imposed by the NB tumor microenvironment, and examine the current state of such therapies in pre-clinical models and clinical trials.

We thank Dr. Kotnik and colleagues for their commentary on the American College of Rheumatology's clinical guidance for pediatric patients with multisystem inflammatory syndrome in children (MIS-C). The use of anticoagulation in this population remains an intensely debated topic with little clinical evidence to guide treatment decisions. For this reason, the task force was only able to achieve consensus on recommending anticoagulation in patients with larger coronary artery aneurysms (CAAs) (z-score >10) and significant cardiac dysfunction (ejection fraction < 35%) based on the well-established risk for thrombosis in patients with these clinical features.(1, 2).

Background The ChAdOx1 nCoV-19 vaccine has been shown to induce Vaccine-induced Immune Thrombotic Thrombocytopenia (VITT), a syndrome that shares clinical features with heparin-induced thrombocytopenia (HIT). The mechanism of thrombocytopenia and thrombosis in these disorders appears to be related to the development of pathologic anti-PF4/heparin antibodies, some of which could activate complement. Interestingly, we and others have found that complement activation is vital when both pediatric and adult patients have severe respiratory illness from SARS-CoV-2 virus (COVID-19) or in the post-infectious multisystem inflammatory syndrome in children (MIS-C). We hypothesized that patients with severe COVID-19 or MIS-C develop similar anti-PF4/heparin antibodies, which lead to endothelial complement activation that drive the inflammatory responses seen in these diseases. Methods Our cohort included 30 pediatric patients with positive SARS-CoV-2 RT-PCRs: 10 each of severe COVID-19 ("Severe", MIS-C, and mild/asymptomatic ("Mild") infection. Using ELISA, we evaluated the levels of antibodies to various platelet-related proteins including PF4, PF4-heparin, and NAP2; in addition, we examined the ability of plasma from each patient to activate complement. The antibody levels were compared to control samples including samples from adult patients with VITT and HIT. Statistical analyses with ANOVA were performed to evaluate differences. Results Patients with MIS-C have a significantly higher anti-PF4 antibody concentration (as measured by mean optical density [OD]) than patients with either mild/asymptomatic disease, or severe COVID-19: Severe 0.5 +/- 0.14; Mild 0.3 +/- 0.12; MIS-C 0.77 +/- 0.35, p=0.003 MIS-C vs. Mild); Similar results were seen for anti-PF4/heparin antibodies: Severe 0.4 +/- 0.14; Mild 0.35 +/- 0.12; MIS-C 0.64 +/- 0.3, p=0.003 MIS-C vs. Mild; p=0.034 MIS-C vs. Severe). These were similar to values obtained for the HIT sample (Figure). Conclusion Patients with MIS-C and severe COVID19 have significant detectable anti-PF4 and PF4/heparin antibodies in contrast to those patients with mild/asymptomatic disease. Our previous studies have shown that patients with MIS-C and COVID-19 have evidence of endovascular complement activation in the form of elevated soluble membrane attack complex (sC5-b9). We have also previously demonstrated that VITT anti-PF4 and anti-PF4/heparin antibodies activate complement and result in endothelial cell activation. These antibodies in pediatric SARS-CoV-2 infection may be involved in the development of more severe disease manifestations. Ongoing investigations will identify if this is due to endothelial complement activation and inflammatory responses that accompany severe disease. This is the first demonstration of the role of anti-PF4 and PF4/heparin antibodies in pediatric SARS-CoV-2. Figure 1 Figure 1. Disclosures Bassiri: Guidepoint Global: Consultancy; Kriya Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company. Teachey: Janssen: Consultancy; NeoImmune Tech: Research Funding; Sobi: Consultancy; BEAM Therapeutics: Consultancy, Research Funding. Lambert: ClinGen, ISTH, ASH, GW University: Honoraria; Rigel: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy; Dova: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Principia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Sysmex: Research Funding; PDSA: Research Funding; Octapharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction: The most common severe toxicity associated with chimeric antigen receptor T-cells targeting CD19 (CART19) is cytokine release syndrome (CRS; PMID: 29972754). Our group and others have published seminal observations on the biology of CRS through cytokine profiling, measuring a small number of analytes (PMID: 27076371, 33434058). Multiple biomarkers including interferon gamma (IFNG), IL-6, and IL-10 have been associated with the development of severe CRS in previous studies (PMID: 33434058). To date, the only biomarker predictive of the development of CRS prior to infusion has been disease burden. To obtain a more robust understanding of CRS biology, we performed comprehensive secretome profiling to measure more than 1400 serum analytes on serial serum samples collected from patients treated with the 41BB-containing CTL019 on two clinical trials. Methods: Serum from patients enrolled on two clinical trials of the CART19 product CTL019 (NCT01626495 & NCT02906371) were obtained serially from pre-infusion to one month post infusion. Patients were categorised as having "minimal" (no CRS, Grade 1, or Grade 2) or "severe" (Grade 3 or 4) CRS. The serum secretome was profiled using the Olink Explore 1536 Analysis platform (Olink, Upsala, Sweden). 1484 proteins were measured from serum via proximity extension assay (PEA) high-multiplex immunoassay. Differential expression analysis, correlation analyses and receiver operating characteristic (ROC) calculations were performed using R (version 4.0.4) in RStudio. Significance was based on a fold change of greater than 2 or less than -2 and a false discovery rate of less than 0.05 calculated using a Benjamini-Hochberg correction. Results: 26 patients (10 NCT01626495 & 16 NCT02906371) were included comprising 128 unique datapoints from baseline to 35 days post-infusion. Thirteen patients had minimal and 13 had severe CRS. Differentially expressed proteins between minimal and severe CRS at the peak timepoint are shown in (A; green represents IFNG responsive proteins). Not surprisingly, proteins involved in IL-6 and IFNG signalling were increased, including biomarkers of hemophagocytic lymphohistiocytosis (HLH) such as VSIG4, CXCL9, CXCL10, CD163. The IL-18 signalling axis was dysregulated at peak CRS in severe patients with markedly elevated IL18 and IL18BP, despite prior reports suggesting IL-18 up-regulation is unique to the late CRS seen with CART22 (PMID: 32925169). Soluble markers of checkpoint inhibition, including soluble PDL1 (CD274) and LAG3 were also highly elevated. Finally, biomarkers of endothelial damage, such as PLAT, TMSB10 and CALCA were significantly elevated in patients with severe CRS. Pathway analysis revealed significant dysregulation in targetable cytokine, chemokine, and signalling pathways (B). A volcano plot of differentially expressed proteins at pre-infusion (C) identified a single protein, MILR1, as a candidate biomarker that was highly differentially expressed in patients who would subsequently develop severe CRS. MILR1 expression decreased over time (D). An ROC of MILR1 as a predictor for development of severe CRS (E) demonstrated pre-infusion elevated MILR1 could accurately predict development of severe CRS (sensitivity 88%, specificity 97%, AUC=0.977). We identified correlates of MILR1 at pre-infusion and found that MILR1 correlated most highly with soluble FLT3 (R=0.86, p Conclusions: With comprehensive secretome profiling we made multiple novel insights into the biology of CRS after CART19 and identified several potentially targetable proteins and pathways that could mitigate severe CRS. Similar secretome profiling in patients who developed neurotoxicity will also be shown. We identified two novel pre-infusion biomarkers that demonstrate significant capacity to predict the development of severe CRS following CART19 infusion. The inverse relationship apparent between FLT3 and FLT3LG that persists over time is an important finding that implies a potential biological role for FLT3/FLT3 ligand in the development of severe CRS. Mechanistic studies exploring the role of MILR1 and FLT3 in the initiation of CRS are ongoing. Figure 1 Figure 1. Disclosures Lambert: Novartis, shionogi, argenx, Rigel, octapharma: Consultancy; Rigel, Novartis, Sysmex, octapharma: Research Funding. Bassiri: Kriya Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company; Guidepoint Global: Consultancy. Levine: Vycellix: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Other: Co-Founder and equity holder; Ori Biotech: Membership on an entity's Board of Directors or advisory committees; Immusoft: Membership on an entity's Board of Directors or advisory committees; Immuneel: Membership on an entity's Board of Directors or advisory committees; Avectas: Membership on an entity's Board of Directors or advisory committees; Akron: Membership on an entity's Board of Directors or advisory committees; In8bio: Membership on an entity's Board of Directors or advisory committees. Maude: Wugen: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding. June: Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Current equity holder in publicly-traded company; Novartis: Patents & Royalties; AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Consultancy. Barrett: Tmunity Therapeutics: Current Employment. Grupp: Novartis, Kite, Vertex, and Servier: Research Funding; Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Teachey: Janssen: Consultancy; NeoImmune Tech: Research Funding; Sobi: Consultancy; BEAM Therapeutics: Consultancy, Research Funding.

Erratum: J Clin Immunol. 2017 Oct;37(7):693-694. doi: 10.1007/s10875-017-0436-0. In today's global economy and affordable vacation travel, it is increasingly important that visitors to another country and their physician be familiar with emerging infections, infections unique to a specific geographic region, and risks related to the process of travel. This is never more important than for patients with primary immunodeficiency disorders (PIDD). A recent review addressing common causes of fever in travelers provides important information for the general population Thwaites and Day (N Engl J Med 376:548-560, 2017). This review covers critical infectious and management concerns specifically related to travel for patients with PIDD. This review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. The organization of this review will address the environment driving emerging infections and several concerns unique to patients with PIDD. The first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with PIDDs. This review does not address most parasitic diseases. Reference tables provide easily accessible information on a broader range of infections than is described in the text.

Introduction: During the Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2), 3 distinct phenotypes have emerged in children. The majority of children have mild or no symptoms. Similar to adults, a minority of children can be severely affected with respiratory distress requiring intensive care. Finally, they may develop a phenomenon presumed unique to children termed Multisystem Inflammatory Syndrome in Children (MIS-C). MIS-C is a hyperinflammatory syndrome characterized by fever and organ dysfunction (particularly cardiac) in the setting of recent COVID-19 infection. Reports from the adult literature have invoked thrombotic microangiopathy (TMA) and complement activation as a potential cause for severe manifestations of COVID-19 (Zhang et al. NEJM. 2020; Campbell et al. Circulation 2020). Soluble C5b9 (sC5b-9), the terminal complement complex, has been implicated as a marker of hematopoietic stem cell transplant associated TMA (HSCT-TMA; Jodele et al. Blood 2014). We sought to elucidate the role of terminal complement activation and TMA in the different pediatric disease phenotypes. Methods: We enrolled children admitted to the Children's Hospital of Philadelphia during the COVID-19 pandemic who had evidence of SARS-CoV-2 infection on reverse transcriptase polymerase chain reaction (RT-PCR) from mucosa, or met clinical criteria for MIS-C. Patients (pts) were classified in to 3 categories: minimal COVID-19 symptoms or incidental finding of SARS-CoV-2 infection, severe COVID-19 requiring ventilatory support, or MIS-C. To investigate the role of TMA in children with COVID-19 we measured sC5b-9 in plasma of pts with the 3 manifestations of SARS-CoV-2, and in healthy controls. sC5b9 was measured in triplicate at two dilutions by ELISA. Proinflammatory cytokines were measured using V-Plex Pro-inflammatory Panel 1 Human Kits and analyzed on a QuickPlex SQ120. P-values were computed using Dunn's multiple comparisons test after Kruskal-Wallis testing. Blood smears were examined by a hematologist and hematopathologist for schistocytes. Results: 50 pts were enrolled on whom complete sC5b9 data were available: minimal COVID-19 (N=18), severe COVID-19 (N=11), and MIS-C (N=21). Plasma was obtained on healthy controls (N=26). The median sC5b9 level in healthy controls (57 ng/mL) differed significantly (p Pro-inflammatory cytokines were measured. Of particular interest to TMA is the neutrophil chemotactic factor IL-8, because of its role as a marker of endothelial damage (Dvorak et al. Front Pediatr 2019). Levels of IL-8 differed significantly between pts with MIS-C (p=0.0166) or pts with severe COVID-19 (p=0.0079), when compared to minimal COVID-19 pts; but not between pts with MIS-C and severe disease (p = 0.99). Blood smears were available on 34 patients. Schistocytes were present in 13/15 (87%) patients with MIS-C, 7/8 (87%) patients with severe COVID-19 and 5/11 (45%) patients with minimal COVID-19 (χ2=6.59, p=0.037). Conclusions: We demonstrate derangements of the final common pathway of complement activation in children with the 3 presentations of SARS-CoV-2. Strikingly, sC5b9s were abnormal even in children with minimal disease or incidental infection. Renal dysfunction correlated with elevations in sC5b9, strengthening the evidence that TMA plays a role in the pathophysiology of SARS-CoV-2 infection. Future work is aimed at further characterizing the role of the complement cascade in the pathogenesis of MIS-C and COVID-19 in children. The long-term complications of endothelial damage and complement activation are unknown and extended follow-up is warranted. Figure 1 Disclosures Diorio: Children's Hospital of Philadelphia: Research Funding; University of Pennsylvania: Research Funding. Lambert:22qSociety: Consultancy; RDMD ITP study: Consultancy; Octapharma: Consultancy, Research Funding; Educational Concepts in Medicine: Consultancy; Shionogi: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Argenix: Consultancy; CdLS Foundation: Consultancy; Sysmex: Research Funding; Dova: Consultancy, Membership on an entity's Board of Directors or advisory committees; Principia: Consultancy, Membership on an entity's Board of Directors or advisory committees; ClinGen: Honoraria; Platelet Disorder Support Association (PDSA): Consultancy; AstraZeneca: Research Funding; Bayer: Consultancy; ITP Australia: Consultancy. Henrickson:Horizon Pharma: Other: ad hoc board meeting. Odom John:Burroughs Wellcome: Research Funding; NIAID: Research Funding. Bassiri:CSL Behring: Other: Spouse receives stocks . Behrens:NIH/NIAID: Research Funding. Teachey:Janssen: Consultancy; Amgen: Consultancy; La Roche: Consultancy; Sobi: Consultancy.

In this chapter we describe the genes responsible for familial or primary hemophagocytic lymphohistiocytosis, and the clinical and laboratory features that are associated with each syndrome. Additionally, we place primary hemophagocytic lymphohistiocytosis into four distinct categories, depending on the presence or absence of hypopigmentation and susceptibility to Epstein–Barr virus-induced lymphoproliferative disease.