Objectives: Arterial catheter-associated proximal ischemic injury (ACAPII) is a rare but serious complication in critically ill pediatric patients who require arterial access. In a cohort of critically ill children with multiple organ dysfunction syndrome (MODS), we investigated proteomic differences in children who developed ACAPII with the aim of identifying mechanistic pathways that may contribute to the pathogenesis of this complication. We hypothesized that the plasma proteome at MODS onset would differ between children who later develop ACAPII and those who do not. Design: Single-center cohort study of pediatric patients with MODS defined by modified Proulx criteria who underwent arterial catheter placement. We obtained plasma samples within 48 hours of MODS onset. Grading of ACAPII was completed by the hospital vascular access team using standardized criteria. Proteomic analysis was performed using Olink proximity extension assay. Gene set enrichment analysis was used to identify mechanistic pathways enriched in ACAPII cases. Setting: Single-center academic PICU. Patients: Pediatric patients with MODS and arterial access at Children's Hospital of Philadelphia from January 2020 to December 2022. Interventions: None. Measurements and main results: Five of 66 (7.6%) MODS patients with arterial access developed ACAPII. Age, severity of illness, and organ dysfunction profiles did not differ between injured and noninjured patients. Six pathways of immune dysregulation, involving Signal Transducer and Activator of Transcription signaling and cytokine-mediated apoptosis, were enriched in patients with ACAPII ( p < 0.0001). Five pathways associated with endothelial dysfunction were also significantly altered in ACAPII patients ( p < 0.0001). Differential expression analysis identified 18 plasma proteins associated with injury after adjustment for age and severity of illness (false discovery rate p < 0.05), supporting the hypothesis that endothelial injury and immune dysregulation may contribute to ACAPII pathogenesis. Conclusions: In a small pediatric cohort, we identified pathways of immune dysregulation and endothelial dysfunction associated with ACAPII. Because these factors precede ACAPII, there may be a window for treatment

Abstract Introduction The prevalence of obesity has increased worldwide over the last three decades, including severe obesity increasing amongst US children in recent years. Unfortunately, most children with obesity become adults with obesity, highlighting the complexities of significant weight loss. To date, the mechanisms by which obesity impacts immune function is not well understood. Here, we assessed the peripheral immune landscape of two distinct groups: 1) healthy children across ages and body mass indices (BMI) at baseline and 2) the impact of weight loss in adolescent patients before and 6-12 months following bariatric surgery. Methods We integrated spectral flow cytometry, in vitro T cell assays, and single-cell RNA-sequencing of PBMCs to evaluate the impact of obesity in otherwise healthy children and the impact of weight loss in adolescents with severe obesity. Results Children living with obesity have dysregulated non-naive CD8+ T cells (Tnn), including increased expression of activation markers (e.g., PD-1, CD69, CD95) and production of effector cytokines (IL-2, IFN-γ, TNF-α, which were significantly associated with BMI. Surgically induced weight loss had relatively minor impacts on immune phenotype and function. Transcriptionally, CD8+ T cells were enriched for cytokine signaling pathways in children with increased BMI, including amplified NF-kB signaling. Conclusion These data demonstrate a pattern of CD8+ T cell dysregulation in obesity that is only somewhat normalized by surgical weight loss. Funding Source Chan Zuckerberg Initiative; Burroughs Wellcome Fund Topic Categories Immune Mechanisms of Human Disease (HUM)

Rationale: Our group recently identified a novel pediatric sepsis endotype associated with dysregulated STAT3 signaling, CD8+ T cell hyperactivation, increased mortality, and higher cumulative organ dysfunction scores. T cell activation can occur via bystander activation, which is associated with off-target tissue injury, or antigen-specific activation, which is associated with pathogen control. We hypothesized that CD8+ T cell hyperactivation within the dysregulated STAT3 signaling endotype would be driven by bystander activation, which could influence treatment strategies. Methods: To analyze T cell activation and T cell receptor (TCR) repertoire within this sepsis endotype, we performed 5’ single-cell RNA sequencing (scRNAseq) and TCR sequencing (TCRseq) on CD45+ lymphocytes from 9 patients with dysregulated STAT3 signaling and 3 age-matched healthy controls. We annotated cell subsets using ScType and performed differential expression analysis using FindMarkers. We assessed CD8+ T cell activation using gene set enrichment analysis (GSEA) and cell-specific ligand-receptor interactions using CellChat. We measured TCR diversity and clonal abundance using scRepertoire. Results: Compared to healthy controls, patients with sepsis have reduced effector CD8+ T cell and increased naïve CD8+ T cell populations (p<0.0001). Perforin and granzyme expression are increased in naïve and effector CD8+ T cells from patients with the dysregulated STAT3 signaling endotype (“STAT3 endotype” patients) compared to healthy controls (all p<0.0001), suggesting increased cytotoxic activity. GSEA of CD8+ T cell subsets revealed increased IFNγ production (NES +2.27 [naïve], +2.46 [effector], both p<0.0001) and cytokine signaling (NES +1.68 [naïve], NES +1.73 [effector], both p<0.01) in STAT3 endotype patients compared to healthy controls, consistent with increased T cell activation. Ligand-receptor interaction analysis demonstrated significant interactions between HLA class I molecules and CD8A in CD8+ T cells from STAT3 endotype patients compared to healthy controls (p<0.01, Figure 1A), suggesting increased TCR signaling. TCR repertoire analysis identified increased clonal diversity in STAT3 endotype patients compared to healthy controls (p<0.0001, Figure 1B) without evidence of clonal expansion (Figure 1C). Within T cell subsets, STAT3 endotype patients demonstrated increased TCR diversity in naïve CD8+ T cells and reduced TCR diversity in effector CD8+ T cells (Figure 1D). Conclusions: Through paired scRNAseq and TCRseq analysis, we identified CD8+ T cell hyperactivation and increased TCR clonal diversity in patients with the dysregulated STAT3 signaling endotype of pediatric sepsis. Polyclonal bystander activation of CD8+ T cells may be a reversible cause of organ failure within this sepsis endotype. Dysregulated STAT3 signaling is a candidate target for precision immunomodulation in pediatric sepsis.

Sepsis is the leading cause of pediatric in-hospital mortality worldwide, driven in part by immune dysregulation. As sepsis biology is marked by immune heterogeneity, precision medicine approaches to identify actionable phenotypes are crucial to improving patient outcomes. Among three molecular subphenotypes we previously identified in children with sepsis, Group C patients have poor clinical outcomes and a unique immune profile involving dysregulated STAT3 signaling. In this analysis, we sought to evaluate cytokine production in CD8+ T cells from pediatric sepsis patients across molecular subphenotypes. We measured T cell cytokine production by flow cytometry in pediatric sepsis samples (n = 17) after 24-hour stimulation with αCD3/αCD28. We calculated absolute and relative change in geometric mean fluorescent intensity from baseline for both conditions for six cytokines: IL-2, IL-13, IL-17, IL-21, TNF-α, and IFN-γ. Samples were acquired via a Cytek Aurora spectral flow cytometer and analyzed in FlowJo and Rstudio. We compared cytokine expression across sepsis subphenotypes by ANOVA. Baseline, unstimulated cytokine production was minimal in all groups. In response to αCD3/αCD28 stimulation, CD8+ T cells from Group C patients demonstrated significantly increased IL-2 production (p = 0.012) compared with Groups A and B, and a trend toward sustained TNF-α production (p = 0.11). Conversely, Group C showed an exaggerated reduction in IL-17 production in response to stimulation (p = 0.03). CD4+ T cells from Group C patients similarly demonstrated significantly increased IL-2 production (p = 0.003) after stimulation compared with Groups A and B, and a similar trend toward sustained TNF-α production (p = 0.11). IFN-γ, IL-13, and IL-21 expression increased with stimulation in all groups but did not vary by subphenotype. Group C patients demonstrate increased pro-inflammatory cytokine production in CD8+ T cells following αCD3/αCD28 stimulation compared with Groups A and B. This dysregulated response to T cell stimulation in the most severe molecular sepsis subphenotype is a potentially reversible cause of organ dysfunction in pediatric patients with sepsis.

STAT1 gain-of-function (GOF) patients experience severe fungal and viral infections and autoimmunity. CD8+ T cells are key mediators of the adaptive immune response by combating infections. CD8+ T cell dysfunction, in turn, contributes to autoimmune pathogenesis and chronic viral infections. We therefore hypothesized that CD8+ T cell dysfunction contributes to STAT1 GOF pathophysiology and sought to identify the underlying mechanisms. We collected high-dimensional immunophenotyping and cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) data on 24 STAT1 GOF patients and age-matched healthy control PBMCs. In addition, T cell function and metabolic data were collected on untreated and in vitro-treated patient and healthy control PBMCs. In patients with STAT1 GOF, CD8+ T cell dysfunction was best defined as reduced production of IFN-γ, TNF-α, and IL-2 upon αCD3/αCD28 stimulation. High-dimensional immunophenotyping identified CD38 as a significantly upregulated activation marker on CD8+ T cells and most immune cell populations, from patients with STAT1 GOF. CD38 expression could be further induced by αCD3/αCD28 stimulation and was correlated with the levels of total STAT1 protein. CD38 is an ectoenzyme that consumes nicotinamide adenine dinucleotide (NAD+), and low NAD+ levels have been linked to immune dysregulation. Our CITE-Seq data confirmed that STAT1 GOF CD8+ T cells have transcriptional evidence of broadly altered NAD+ metabolism, with both NAD-consuming enzymes such as CD38 and PARP9 as well as NAD+ salvage pathway proteins such as NAMPT being increased. Using an NAD+ enzymatic assay, we confirmed that NAD+ levels were indeed reduced in STAT1 GOF CD8+ T cells while the NADH levels were comparable. By increasing NAD+ levels in patient PBMCs, it was possible to partially normalize CD8+ T cell function in CD8+ T cells from patients with STAT1 GOF. Finally, STAT1 GOF patients receiving JAK inhibitors show reduced CD38 RNA and protein expression and reduced PARP9 and NAMPT transcription. Elevated CD38 expression and reduced NAD+ levels contribute to CD8+ T cell dysfunction in CD8+ T cells from patients with STAT1 CD8+ T cells, which can be partially normalized by providing increased NAD+ levels.

Sepsis is a leading cause of morbidity and mortality in critically ill children, yet heterogeneity in immune responses complicates the development of targeted therapies. Although immune dysregulation is associated with poor outcomes in sepsis, it remains unclear which host immune factors contribute causally to sepsis morbidity and mortality. To address this gap, we integrated deep immune phenotyping, plasma proteomics, single-cell transcriptomics, and phosphoflow cytometry in a prospective cohort of 88 critically ill children to elucidate the immunologic mechanisms which underly disease heterogeneity. Unsupervised clustering of plasma cytokines identified three immunologic subgroups, including a high-severity group ("Group C") characterized by marked hypercytokinemia, driven primarily by IL-6 and IFN-γ. Group C exhibited distinct alterations in immune cell frequency and activation status, along with a strong association between hyperinflammatory signaling and lymphocyte dysfunction. Single-cell RNA sequencing revealed transcriptional signatures of T cell activation and metabolic stress, and identified widespread suppression of a lymphoid protective gene program across CD8⁺ T cell subsets. In the setting of increased expression of activation markers, T cell receptor repertoire analysis revealed no dominant clonotypes, consistent with a bystander mechanism of T cell activation. Using phosphoflow cytometry, we demonstrated baseline hyperactivation of STAT1 and STAT3 in CD8⁺ T cells from patients in Group C, and these cells failed to respond to aCD3/aCD28 stimulation. Together, these findings define IL-6/IFN-γ-driven T-cell dysfunction as a distinct endotype of immune dysregulation in pediatric sepsis, highlighting the JAK/STAT axis as a potential future target for immunomodulatory therapy.

Abstract Regulation of T cell signaling is paramount to mount an appropriate immune response and protect the host from pathophysiology. Here, we study the impact on CD8+ T cells in monogenic inborn error of immunity patients exhibiting dysregulated signal 3 (cytokine signaling) pathways resulting from STAT3 gain- or loss-of-function variants (STAT3 GOF and STAT3 LOF). Using high dimensional immune profiling, we demonstrate increased expression of the ectoenzyme, CD39, which hydrolyzes extracellular ATP, on STAT3 GOF patient CD8+ T cells. Moreover, CD8+ T cells from two mouse models of STAT3 GOF (STAT3+/G421R and NOD-STAT3+/K392R) recapitulated this increase in CD39. In vitro, STAT3-activating cytokines (e.g., IL-6, IL-21, IL-27, IL-10) augment CD39 induction in a TCR-dependent manner in healthy donor and in STAT3 GOF CD8+ T cells. In contrast, STAT3 LOF patients show diminished CD39 induction. We find a positive correlation between CD39 induction and phosphorylated STAT3 levels while STAT3 inhibition prevents this cytokine-driven augmentation of CD39. Finally, acutely activated, and sorted CD39+CD8+ T cells from healthy donors inhibit bystander CD8+ T cell cytokine production (e.g., IFN-g). Pharmacologic inhibition of CD39 enzymatic activity or genetic ablation via CRISPR/Cas9 editing reversed decreases in cytokine production. Together, our data demonstrate a mechanistic connection between amplified STAT3 signaling and CD39 levels which is capable of impairing CD8+ T cell function.

Lindell, Robert; Sayed, Samir; Henrickson, Sarah; Gollomp, Kandace; Grundmeier, Robert; Weiss, Scott; Alpern, Elizabeth; Nelson Sanchez-Pinto, L.; Balamuth, Frances Author Information

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre–α T cell receptor (pre-TCRα) expression. Low circulating naive αβ T cell counts at birth persisted over time, with normal memory αβ and high γδ T cell counts. Their TCRα repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue αβ T cell development. Only a minority of these individuals were sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naive αβ T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αβ T cells, autoimmune conditions were more frequent in these patients compared with the general population.

Abstract The increasing prevalence of childhood obesity is an urgent public health crisis. Children with obesity (≥ 95th percentile BMI for age and sex) are at an increased risk for developing asthma as well as cardiometabolic and autoimmune disorders. However, the mechanisms by which excess weight impacts immune function in children is incompletely understood. To address this gap in knowledge, we assessed changes in immune cell function in obese children before and after bariatric surgery, a procedure used to facilitate weight loss in patients with severe obesity. We integrated high dimensional spectral flow cytometry, in vitro functional assays, and single-cell RNA-sequencing of intra-operative visceral adipose tissue and PBMCs to find that CD8+ T cells from obese children, compared to age-matched controls, had increased expression of TOX and inhibitory receptors (PD-1, LAG-3). This dysfunctional phenotype was persistent 6-18 months after surgery, despite significant weight loss (average change in BMI of 17%). Moreover, baseline CD8+ T cells from obese patients exhibit impaired proliferation and altered metabolic profiles following TCR stimulation. Future work will determine functional differences in the peripheral T cells of bariatric surgery patients at baseline compared to 6-18 months post-surgery and assess the local immune landscape within visceral adipose depots from obese compared to non-obese children.

Signal transduction downstream of activating stimuli controls CD8+ T cell biology, however these external inputs can become uncoupled from transcriptional regulation in Primary Immune Regulatory Disorders (PIRDs). Gain-of-function (GOF) variants in STAT3 amplify cytokine signaling and cause a severe PIRD characterized by early onset autoimmunity, lymphoproliferation, recurrent infections, and immune dysregulation. In both primary human and mouse models of STAT3 GOF, CD8+ T cells have been implicated as pathogenic drivers of autoimmunity. The molecular mechanisms by which STAT3 GOF variants drive this pathology remain unclear. We found that naive CD8+ T cells have an increased capacity for IFN-g and TNF-a secretion. Given this dysregulation of CD8+ T cell function, we evaluated changes in immunoregulatory pathways and found evidence of dysregulated purinergic signaling via high dimensional immune profiling, single-cell RNA sequencing, and functional assessment. Specifically, while expression of CD39, which transforms ATP to AMP, was increased on CD8+ T cells from patients with STAT3 GOF, downstream purinergic family members, CD73 and the adenosine receptor, A2AR, were downregulated, impairing the potential to produce or sense inhibitory adenosine. Patients with STAT3 GOF can be clinically treated with JAK inhibitors, and this partially normalized naive CD8+ T cell dysregulation, including aberrant cytokine production. The extent of normalization scaled with normalization of CD73 and A2AR. This suggests that a dysregulated purinergic signaling axis plays an important role in CD8+ T cell dysregulation in STAT3 GOF, which may have implications for other inflammatory disorders with amplified STAT signaling.

Background: Primary Immune Regulatory Disorders (PIRDs) are a complex and challenging-to-treat subset of Inborn Errors of Immunity (IEI), which are characterized by immune dysregulation leading to recurrent infections, lymphoproliferation, and autoimmunity including refractory cytopenias. Since many ultra-rare monogenic PIRDs exist, it is not feasible to design targeted therapies for each. An alternate strategy is to identify shared aspects of T cell dysfunction and strategies targeting them. We have focused our studies on PIRDs that chronically amplify T cell receptor (TCR) signaling, mimicking chronic infection. Under conditions of chronic inflammation and antigen presentation, seen during chronic infections, CD8 T cell exhaustion (Tex) can result and is characterized by increased inhibitory receptor expression, altered transcriptional networks, epigenetic poise, and impaired T cell functions including cytokine production. Here, we have evaluated CD8 T cell dysfunction in PIRDs, including the potential for Tex. Methods: Deep immune phenotyping and T cell functional analyses were performed using CyTOF and spectral flow cytometry, in addition to single cell RNA-sequencing and CITE-seq from untreated PIRD and healthy control PBMCs. Finally, CRISPR/Cas9 editing in healthy control CD8 T cells was used to create a cellular disease model. Results: We identified a Tex-like process in activated PI3 kinase delta syndrome (APDS), CTLA-4 haploinsufficiency, and Ras-associated Autoimmune Leukoproliferative Disease (RALD), which share increased mTOR activation. We identified increased PD-1, CD39, TIGIT and TOX expression on CD8 T cells consistent with a Tex-like phenotype. We also found impaired CD8 T cell cytokine and proliferation consistent with Tex. Lastly, a cellular model of CTLA-4 haploinsufficiency was created for perturbation studies to evaluate best available therapies and target novel therapeutics in these rare disorders. Conclusion: By identifying shared patterns of CD8 T cell dysfunction in these ultra-rare disorders, we may both identify novel therapeutic strategies and increase our understanding of CD8 T cell function, including cytokine production.

It is still unknown whether primary immune regulatory disorders (PIRD) due to genetic variants that amplify T cell receptor and/or inflammatory cytokine signaling yield T cell exhaustion. Here, we investigate the impact of dysregulated cytokine signaling on CD8 +T cells in STAT3 gain-of-function (STAT3 GOF) patients. Immune profiling via 43-parameter CyTOF reveals altered CD8 +T cell differentiation, including loss of naïve cells, increased frequency of effector memory and T EMRAcells, and downregulation of markers of stemness (i.e., TCF-1 and CD127) in STAT3 GOF. Additionally, STAT3 GOF CD8 +T cells demonstrate decreased IL-2 production and proliferation. We identify increased expression of the ecto-enzyme, CD39, which has been reported on exhausted cells and hydrolyzes extracellular ATP, on CD8 +T cells from STAT3 GOF patients and in a mouse model of STAT3 GOF. To identify the signals regulating CD39, healthy donor CD8 +T cells were cultured with STAT3-activating cytokines alone but did not upregulate CD39. However, TCR engagement led to robust CD39 expression which was further augmented with STAT3-activating cytokines. Specifically, CD39 upregulation occurs in pSTAT3 Y705+cells. Finally, sorted CD39 +CD8 +T cells hydrolyzed ATP and suppressed effector CD8 +T cell proliferation compared to CD39 −CD8 +T cells. These data suggest that STAT3 signaling may play a role in regulating CD39 levels and thus contribute to CD8 +T cell exhaustion, which will be explored in future work. Understanding how dysregulated STAT3 signaling impacts T cell function in these rare patients can improve our understanding of this complex disease and may yield insights into shared aspects of T cell dysfunction found in more common inflammatory diseases. HHMI Gilliam Fellowship for Advanced Studies (GT15736) Penn Presidential Fellowship PIDTC (Henrickson Lab) IDF (Henrickson Lab) NIH NIAD K08AI135091 (Henrickson Lab) Burroughs Wellcome Fund CAMS (Henrickson Lab)

STAT1 has a central role in relaying signals from type I, II and III interferons (IFN), thus contributing to the antiviral immune response. Working in tandem, CD8+ T cells (CD8T) are key mediators of the adaptive immune response to viral infections. In patients with STAT1 gain of function (GOF) syndrome there is an increase in viral infections, as part of their immune dysregulatory phenotype. However, there is currently a limited understanding of how increased STAT1 signaling might alter CD8T function, potentially impairing anti-viral mechanisms. We thus aim to define the extent and nature of CD8T dysfunction and underlying mechanisms in STAT1 GOF patients. We collected multi-modal high-dimensional immune profiling and functional data including phosphoflow on peripheral blood mononuclear cells (PBMCs) from 24 STAT1GOF patients and age matched controls. 7 Aicardi-Goutières-Syndrome (AGS) patients were included as a comparison population. These cohorts allow us to differentiate the effects of increased type I IFN signaling vs chronic STAT1 activity on CD8T function. STAT1 GOF CD8T showed signs of immune dysregulation including reduced production of IFN-γ and TNF-α upon phorbol-myristate-acetate (PMA) and ionomycin stimulation. High-dimensional immune profiling revealed many activation markers altered on STAT1 GOF CD8T. CD38 was overall significantly elevated, with CD4T and CD8T most affected in STAT1GOF patients. This is also seen to a lesser extent in AGS patients. CD38 was most efficiently induced by T cell receptor (TCR) stimulation in combination with type I interferon in both healthy control and STAT1GOF CD8 T in vitro. Upon further analysis it was the STAT1hi CD8T, known to be elevated in STAT1GOF, that most dynamically upregulated CD38. Conclusion and Outlook: In STAT1 GOF, a model of chronic STAT1 signaling, we found increased expression of CD38 on CD8T. CD38 acts as an ectoenzyme that consumes nicotinamide adenine dinucleotide (NAD+). Elevated CD38 and low NAD+ have been associated with states of immune dysregulation. We thus suggest CD38 as a promising candidate present on STAT1GOF CD8T that might alter NAD+ levels. This could be a potential novel mechanism underlying the immune dysregulation present in STAT1GOF patients.

A key outcome of chronic antigenic stimulation in the setting of sustained inflammation is the development of CD8+ T cell exhaustion. However, the relative roles of inflammation and antigen stimulation on T cell dysfunction remain unclear. Inborn errors of immunity (IEIs) provide an opportunity to study the effect of genetic alterations in signaling pathways on immune function. Here, we focus on an IEI resulting from genetically amplified cytokine signaling: gain-of-function mutations in STAT3 (STAT3 GOF). STAT3 signals downstream of multiple inflammatory cytokines (e.g., IL-6 and IL-27) and we used STAT3 GOF as a genetic mimic of chronic inflammation. To assess how amplified STAT3 signaling impacts immune cell subset distribution and activation status, PBMCs from STAT3 GOF patients and age-matched healthy controls were profiled via mass cytometry (CyTOF). This revealed altered states of CD8+ T cell activation including increased expression of immunoregulatory receptors (e.g., PD-1 and CD39). STAT3 GOF CD8+ T cells also showed impaired cytokine production and reduced proliferative capacity. Bulk RNA sequencing of naïve STAT3 GOF CD8+ T cells identified an exhausted-like transcriptional profile. Quantitation of metabolic function shows alterations in glucose use and oxidative phosphorylation in STAT3 GOF. Our studies suggest that inflammation alone may directly impair CD8+ T cell function. Understanding the mechanisms by which inflammation impacts T cell function may identify components of T cell dysregulation that can be targeted therapeutically in these rare patients, as well as those with more common inflammatory diseases (e.g., obesity) with shared aspects of T cell dysfunction. Supported by CHOP Research Institute, The Primary Immune Deficiency Treatment Consortium (PIDTC) and Immune Deficiency Foundation (IDF).