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.

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.

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.

The ability of most patients with selective immunoglobulin A (IgA) deficiency (SIgAD) to remain apparently healthy has been a persistent clinical conundrum. Compensatory mechanisms, including IgM, have been proposed, yet it remains unclear how secretory IgA and IgM work together in the mucosal system and, on a larger scale, whether the systemic and mucosal anti-commensal responses are redundant or have unique features. To address this gap in knowledge, we developed an integrated host-commensal approach combining microbial flow cytometry and metagenomic sequencing (mFLOW-Seq) to comprehensively define which microbes induce mucosal and systemic antibodies. We coupled this approach with high-dimensional immune profiling to study a cohort of pediatric patients with SIgAD and household control siblings. We found that mucosal and systemic antibody networks cooperate to maintain homeostasis by targeting a common subset of commensal microbes. In IgA-deficiency, we find increased translocation of specific bacterial taxa associated with elevated levels of systemic IgG targeting fecal microbiota. Associated features of immune system dysregulation in IgA-deficient mice and humans included elevated levels of inflammatory cytokines, enhanced follicular CD4 T helper cell frequency and activation, and an altered CD8 T cell activation state. Although SIgAD is clinically defined by the absence of serum IgA, the symptomatology and immune dysregulation were concentrated in the SIgAD participants who were also fecal IgA deficient. These findings reveal that mucosal IgA deficiency leads to aberrant systemic exposures and immune responses to commensal microbes, which increase the likelihood of humoral and cellular immune dysregulation and symptomatic disease in patients with IgA deficiency.

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).

Pediatric obese atopic asthma (OA) is a complex and poorly understood disease at the intersection of two of the most common chronic inflammatory diseases of childhood. Pediatric OA patients often experience severe asthma exacerbations caused by respiratory viral infections. However, the immunological mechanisms by which obesity modifies immune function in asthma remain poorly understood. Using high dimensional immune profiling, we have demonstrated CD8 T cells from pediatric OA patients have an exhausted-like immunophenotype compared to non-obese asthmatics. To more deeply characterize the role of CD8 T cell dysfunction in OA, we developed a mouse model of OA using high fat diet (HFD) to induce obesity and house dust mite (HDM) to induce asthma. We found that non-naïve CD8 T cells isolated from the lungs of OA mice demonstrate increased expression of multiple activation and inhibitory receptors (e.g., PD-1, CD39 and Tim3) as well as TOX, all consistent with T cell exhaustion, compared to healthy controls. We hypothesize the exhausted-like state of CD8 T cells in OA impairs anti-viral responses, thereby increasing severity of asthma exacerbation during respiratory viral infections. Ongoing work is testing antigen specific responses to influenza infection. Overall, this reveals a previously unrecognized role for exhausted-like CD8 T cell responses in mediating the immunopathogenesis of pediatric obese asthma. This work was supported by the Translational Research Training Program in Environmental Health Sciences Pre-Doctoral Fellowship to C.A.H. and K08-AI135091 to S.E.H.

Immunoglobulin A (IgA) is a fundamental component of mucosal immunity, but only ∼1/3 of patients with selective IgA deficiency (SIgAD) are symptomatic with increased rates of allergy, infection and/or autoimmunity. We cannot yet explain or predict the variability in the clinical phenotype so we sought to investigate whether IgA impacts access of gut microbes to the systemic immune compartment and immune function. We recruited a cohort of pediatric SIgAD patients, with unaffected sibling controls and collected blood and stool. We performed immune profiling (flow cytometry and CyTOF) and microbial flow cytometry (mFlow) and metagenomic sequencing. In mFlow, patient's serum is added to their stool microbes and Ig binding to these microbes is measured by flow cytometry (to measure anti-commensal responses). Stool microbes can then be sorted based on whether bound IgA, IgM and/or IgG and sequenced (in addition to bulk stool metagenomic sequencing). Patients who were deficient in both serum and stool IgA had fewer IgA+ memory B cells and IgG+ B cells. Serum cytokine analysis demonstrated a broad increase in inflammatory cytokines in SIgAD patients. SIgAD patients lack IgA bound microbes and have higher frequency of IgG bound microbes. SIgAD has significant impacts on immune phenotype (B cell differentiation, inflammatory cytokine profile and T cell activation) and the access of commensal gut microbes to the systemic immune compartment. We are currently linking these alterations to the diverse phenotypes of IgA deficiency in humans and mice.

Dysregulated cytokine signaling due to alterations in STAT1 and STAT3 signaling may be perceived by T cells as amplified or skewed inflammation, impairing tolerance to self and our protective responses to pathogens and malignancy. STAT1 and STAT3 GOF mutations impersonate unchecked Type I and II Interferon signaling and chronic IL-6 signaling, respectively, yielding immune dysregulation whereas LOF mutations yield pathogen susceptibility. Here, we focus on quantifying the immunometabolic mechanisms underlying T cell dysregulation as a result of altered STAT signaling. Patient and age-matched healthy control PBMCs were profiled via 43-parameter mass cytometry (CyTOF). Intracellular cytokine expression was measured by flow cytometry following PMA/Ionomycin stimulation. Cell energy metabolic flux was assessed by Seahorse. Analysis of altered transcriptional networks in non-naïve CD4+ and CD8+ T cells is ongoing. Immune profiling of a cohort of STAT1 and STAT3 GOF and LOF patients demonstrates imbalances in the CD4 T helper cell subsets and exhaustion-like dysregulation of activation markers (e.g. Ki67 and Granzyme B) and inhibitory receptors (e.g. CD39, PD-1, CTLA-4, TIGIT) in non-naive CD8 T cells. In addition, both CD4 and CD8 non-naive T cells had altered cytokine production (e.g. IL-2, IFN-gamma). Immunometabolic dysregulation as a result of altered STAT signaling affects both T cell phenotype and function, yielding potential novel therapeutic targets for our rare patients as well as deeper understanding of these fundamental cytokine signaling pathways. We will pursue these hypotheses in mouse models and in vitro human cellular studies.

Primary Immune Regulatory Disorders (PIRD), with their complexity of increased prevalence of autoimmunity and malignancy, as well increased infection susceptibility, are increasingly appreciated as a significant subset of Inborn Errors of Immunity (IEI). A subset of PIRD, including activated PI3 kinase delta syndrome (APDS), may mimic chronic infection due to their ongoing activating signaling downstream of the TCR and costimulatory pathways. This mimicry could yield an exhaustion-like immunometabolic dysregulation of T cell activation and function which could be targetable therapeutically by existing or novel treatments. Quantifying these alterations could potentially improve clinical care and our understanding of this key pathway. A small cohort of APDS patients pre- and post-leniolisib therapy and healthy control (HC) PBMCs were analyzed via mass cytometry (CyTOF) and via RNA sequencing of sorted naïve and non-naïve CD4 and CD8 T cells. Immune profiling of APDS patients at baseline compared to HC demonstrated alterations in T cell activation markers, inhibitory receptors and transcription factors associated with T cell activation and exhaustion. Alterations in T cell activation were supported by our transcriptome analysis using GSEA and pySCENIC. These alterations were only partially corrected by targeted leniolisib therapy. Overactive PI3K signaling in APDS may mimic chronic infection and the resulting exhaustion-like immune phenotype may potentially be secondary to immunometabolic dysfunction. Inability to completely reverse the markers of immune dysregulation in our small cohort calls for study of additional patients, longer follow up and exploring the role of earlier and/or additional therapies.

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 spikespecific 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.

Childhood asthma and obesity are two of the most common chronic diseases worldwide. As body mass increases, there is an increase in asthma prevalence, supporting a possible pathophysiological link. While the mechanism of that link remains unclear, immunometabolic dysfunction is increasingly appreciated as a key mechanism in immunological disease and hereperwe tested that hypothesis. Prospective immunometabolic study of pediatric asthma and obesity with recruitment of obese asthmatics (OA), non-obese asthmatics (A), obese non-asthmatics (O) and healthy controls (HC). Asthmatics were recruited from our Allergy clinic with deep clinical metadata. To assess the underlying mechanisms of immune dysfunction, we used peripheral blood mass cytometry (CyTOF), flow cytometry, serum metabolomics and cytokine analysis and high dimensional systems immunology analytics. A second cohort of asthmatics (OA and A) was assessed as well. Human in vitro culture of PBMCs and a mouse model of obese asthma were then used to test hypothesized mechanisms of T cell dysfunction from these studies. Pediatric atopic OA demonstrated alterations in T cell differentiation including increased type 2 immunity and T cell exhaustion. OA also demonstrated altered serum metabolites including glutamate and acetate, which were shown to underlie some of the immune dysfunction using in vitro and in vivo studies. We have identified novel insights into the mechanistic links between metabolic disturbances and immune dysfunction in obesity and asthma. We are further extending mechanistic testing in mouse models of obese asthma and in vitro in human studies to move towards identification of novel therapeutic strategies targeting these mechanisms.

Selective IgA deficiency (sIgAD) is the most common primary immune deficiency. Symptomatic patients can experience increased atopy, recurrent infections or autoimmunity, though patients are frequently asymptomatic. However, we lack prognostic markers. Since IgA promotes homeostasis with commensal microbes, we investigated whether sIgAD impaired commensal microbe compartmentalization and altered systemic immune responses Blood and fecal samples were collected from 15 pairs of pediatric sIgAD patients and IgA sufficient siblings. Deep immunoprofiling using flow cytometry, CyTOF, cytokine analysis and ELISAs for Ig binding to fecal microbes was combined with metagenomic analysis of fecal microbiomes and microbial flow cytometry (mFLOW) of the IgA, IgG and IgM bound fecal microbiomes. mFLOW was performed by applying patient's serum antibodies to their fecal microbes, assessing binding of immunoglobulin isotypes and metagenomic sequencing of Ig-bound microbiomes. Higher frequency of fecal microbes targeted by serum IgG (24.3% vs. 14.3%) and elevated serum sCD14 in sIgAD, indicating enhanced systemic immune response against commensals. We identified microbes enriched in sIgAD patients and microbes that selectively induce a systemic IgG response in sIgAD. Unexpectedly, 15% of sIgAD patients had normal stool IgA, and those deficient in serum and stool IgA had fewer IgA+ memory B cells. We built a controlled pediatric cohort to investigate the effect of sIgAD on systemic immune responses to commensal organisms and used systems immunology strategies to analyze this multimodal dataset. sIgAD has significant impacts on immunophenotype and access of the systemic immune response to commensal gut microbes. These findings provide novel strategies for developing prognostic markers.