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Signal transducer and activator of transcription 3 (STAT3) is a key transcription factor involved in regulation of immune cell activation and differentiation. Recent discoveries highlight the role of germline activating STAT3 mutations in inborn errors of immunity characterized by early-onset multi-organ autoimmunity and lymphoproliferation. Much progress has been made in defining the clinical spectrum of STAT3 GOF disease and unraveling the molecular and cellular mechanisms underlying this disease. In this review, we summarize our current understanding of the disease and discuss the clinical phenotype, diagnostic approach, cellular and molecular effects of STAT3 GOF mutations and therapeutic concepts for these patients.

Non-canonical NF-κB-pathway signaling is integral in immunoregulation. Heterozygous mutations in have recently been established as a molecular cause of common variable immunodeficiency (CVID) and DAVID-syndrome, a rare condition combining deficiency of anterior pituitary hormone with CVID. Here, we investigate 15 previously unreported patients with primary immunodeficiency (PID) from eleven unrelated families with heterozygous -mutations including eight patients with the common p.Arg853 nonsense mutation and five patients harboring unique novel C-terminal truncating mutations. In addition, we describe the clinical phenotype of two patients with proximal truncating mutations. Cohort analysis extended to all 35 previously published -cases revealed occurrence of early-onset PID in 46/50 patients (mean age of onset 5.9 years, median 4.0 years). ACTH-deficiency occurred in 44%. Three mutation carriers have deceased, four developed malignancies. Only two mutation carriers were clinically asymptomatic. In contrast to typical CVID, most patients suffered from early-onset and severe disease manifestations, including clinical signs of T cell dysfunction e.g., chronic-viral or opportunistic infections. In addition, 80% of patients suffered from (predominately T cell mediated) autoimmune (AI) phenomena (alopecia > various lymphocytic organ-infiltration > diarrhea > arthritis > AI-cytopenia). Unlike in other forms of CVID, auto-antibodies or lymphoproliferation were not common hallmarks of disease. Immunophenotyping showed largely normal or even increased quantities of naïve and memory CD4 or CD8 T-cells and normal T-cell proliferation. NK-cell number and function were also normal. In contrast, impaired B-cell differentiation and hypogammaglobinemia were consistent features of -associated disease. In addition, an array of lymphocyte subpopulations, such as regulatory T cell, Th17-, cTFH-, NKT-, and MAIT-cell numbers were decreased. We conclude that heterozygous damaging mutations in represent a distinct PID entity exceeding the usual clinical spectrum of CVID. Impairment of the non-canonical NF-κB pathways affects function and differentiation of numerous lymphocyte-subpopulations and thus causes a heterogeneous, more severe form of PID phenotype with early-onset. Further characteristic features are multifaceted, primarily T cell-mediated autoimmunity, such as alopecia, lymphocytic organ infiltration, and in addition frequently ACTH-deficiency.

Information regarding the prevalence of infectious diseases (IDs) in child and adolescent refugees in Europe is scarce. Here, we evaluate a standardized ID screening protocol in a cohort of unaccompanied refugee minors (URMs) in a municipal region of southwest Germany. From January 2016 to December 2017, we employed a structured questionnaire to screen a cohort of 890 URMs. Collecting sociodemographic information and medical history, we also performed a standardized diagnostics panel, including complete blood count, urine status, microbial stool testing, tuberculosis (TB) screening, and serologies for hepatitis B virus (HBV) and human immunodeficiency virus (HIV). The mean age was 16.2 years; 94.0% were male, and 93.6% originated from an African country. The most common health complaints were dental problems (66.0%). The single most frequent ID was scabies (14.2%). Of the 776 URMs originating from high-prevalence countries, 7.7% and 0.4% tested positive for HBV and HIV, respectively. Nineteen pathogens were detected in a total of 119 stool samples (16.0% positivity), with intestinal schistosomiasis being the most frequent pathogen (6.7%). Blood eosinophilia proved to be a nonspecific criterion for the detection of parasitic infections. Active pulmonary TB was identified in 1.7% of URMs screened. Of note, clinical warning symptoms (fever, cough >2 weeks, and weight loss) were insensitive parameters for the identification of patients with active TB. Study limitations include the possibility of an incomplete eosinophilia workup (as no parasite serologies or malaria diagnostics were performed), as well as the inherent selection bias in our cohort because refugee populations differ across Europe. Our study found that standardized ID screening in a URM cohort was practicable and helped collection of relevant patient data in a thorough and time-effective manner. However, screening practices need to be ameliorated, especially in relation to testing for parasitic infections. Most importantly, we found that only a minority of infections were able to be detected clinically. This underscores the importance of active surveillance of IDs among refugees.

Severe aplastic anemia (SAA) is a life-threatening bone marrow failure syndrome whose development can be triggered by environmental, autoimmune, and/or genetic factors. The latter comprises germ line pathogenic variants in genes that bring about habitually predisposing syndromes as well as immune deficiencies that do so only occasionally. One of these disorders is the autosomal dominant form of chronic mucocutaneous candidiasis (CMC), which is defined by germ line STAT1 gain-of-function (GOF) pathogenic variants. The resultant overexpression and constitutive activation of STAT1 dysregulate the Janus kinase/signal transducer and activator of transcription 1 (STAT) signaling pathway, which normally organizes the development and proper interaction of different components of the immunologic and hematopoietic system. Although SAA is an extremely rare complication in this disorder, it gained a more widespread interest when it became clear that the underlying causative pathomechanism may, in a similar fashion, also be instrumental in at least some of the idiopathic SAA cases. Based on these premises, we present herein what is the historically most likely first cord blood–transplanted SAA case in a CMC family with a documented STAT1 GOF pathogenic variant. In addition, we recapitulate the characteristics of the six CMC SAA cases that have been reported so far and discuss the significance of STAT1 GOF pathogenic variants and other STAT1 signaling derangements in the context of these specific types of bone marrow failure syndromes. Because a constitutively activated STAT1 signaling, be it driven by STAT1 GOF germ line pathogenic variants or any other pathogenic variant-independent events, is apparently important for initiating and maintaining the SAA disease process, we propose to acknowledge that SAA is one of the definite disease manifestations in STAT1 -mutated CMC cases. For the same reason, we deem it necessary to also incorporate molecular and functional analyses of STAT1 into the diagnostic work-up of SAA cases.

BackgroundCD8+ T cells are a critical component of the immune response to intracellular infections and malignancies. Recently, tissue-resident CD8+ memory cells (Trm) have been shown to provide a first line of defense against reinfection at barrier tissues such as the intestine. However, the transcriptional networks regulating tissue-adaptation processes are incompletely understood. Here, we sought to define the transcriptional orchestration of small intestinal Trm cells.MethodsTo identify gene-expression and genome-accessibility changes that arise in Trm cells in response to acute LCMV infection, we used scRNA-seq and ATAC-seq of cells from small intestine (SI), kidney, liver, salivary glands, as well as the spleen and blood. To assess the importance of the transcription factor Hic1 for the differentiation of intestinal Trm cells we used short-hairpin RNAs, inducible genetic deletion mouse models, pharmacological inhibition and Cut&Run sequencing.ResultsRNA sequencing of tissue Trm reveals shared and tissue-specific gene-expression programs, along with intra-tissue heterogeneity of Trm populations. Using transcriptional-regulatory networks, we identify the transcription factor Hic1 as a regulator for intestinal Trm differentiation. We observe that knockdown of Hic1 hinders Trm formation in the SI epithelium in response to both LCMV Armstrong and Listeria monocytogenes infection. In contrast, overexpression of Hic1 promotes Trm formation in the SI upon infection and provides protection upon reinfection. Consistent with Hic1 being a transcriptional repressor, we observe that Hic1 binding sites at genes downregulated by SI Trm lose accessibility in SI Trm. However, Hic1 enhances P2rx7 expression, an eATP receptor previously shown to be important for memory T cell differentiation and Trm homeostasis, by directly binding to its promoter, suggesting that Hic1 acts as both an activator and a repressor during SI Trm formation. Surprisingly, deletion of Hic1 after establishment of intestinal Trm cells does not reduce Trm cell numbers. In line with that, retinoic acid (RA) induces expression of Hic1 and pharmacological blockade of the RA receptor alpha reduces Trm formation, but does not impair Trm maintenance. Further, we find that SI Trm are genetically imprinted to reenter the intestine upon secondary infection, whereas this tissue-specific bias was not observed for liver and kidney Trm cells.ConclusionsIn summary, our work highlights the broad transcriptional adaptations of Trm to a range of tissue environments and highlights the importance of Hic1 as a regulator for SI Trm cell differentiation, which may be used as a framework for identifying targets that influence tissue-specific Trm populations in therapeutic contexts.

BackgroundIn malignancies, CD8+ tumor-infiltrating lymphocytes (TIL) can target tumor cells, but often fail to cure disease due to chronic TIL activation in the tissue’s immunosuppressive tumor microenvironment, resulting in differentiation into an exhausted T cell state. In healthy tissues, T cells differentiate into tissue-resident memory (TRM) in response to infection, and after clearance of antigen can remain to survey and protect from reinfection. When TRM-like TIL are found in cancerous tissue, improved responses to immunotherapy and better patient outcomes are observed; therefore, understanding the connection between exhausted TIL and TRM can inform efforts to manipulate T cell fates towards TRM-like TIL to benefit cancer immunotherapy.MethodsTo better understand the relationship among TRM-like TIL, exhaustion states, and TRM, we directly compared TRM from acute viral infection and exhausted TIL from tumors to find proteomic and transcriptional differences between these distinct T cell states. Focusing on genes highly expressed by TRM that may mediate their enhanced functions in tissues, we looked at genes that became downregulated as T cells transitioned to terminal exhaustion. This approach identified numerous genes related to protein homeostasis (proteostasis), including multiple under-characterized E3 ubiquitin ligases.ResultsProtein regulation by the ubiquitin-proteosome system is an essential biological process, crucial for cell differentiation, and by investigating proteostasis in TIL, we found that exhausted T cells have an excess of misfolded proteins accumulated in their cytosol. CRISPR knockout studies in acute infection showed that loss of our E3 ubiquitin ligases decreased T cell stemness in tissues, and accelerated differentiation to their terminal fate via decreased ubiquitin-mediated proteasomal protein recycling. When we enforced expression of these ligases in tumor-specific T cells in cancer, we found the transduced T cells showed a decrease in cytosolic misfolded proteins and showed enhanced anti-tumor function: increased accumulation in the TME, upregulation of TRM markers, and increased IFN-gamma production, which resulted in better tumor control and improved mouse survival.ConclusionsThese data highlight an underappreciated relationship between proteostasis and T cell stemness, and provide novel avenues of immunotherapeutic approaches for cancer.Ethics ApprovalThe study was approved by UCSD’s Institutional Animal Care and Use Committee, protocol number S04105.

BackgroundCD8+ naive T cells undergo clonal expansion upon antigen encounter, generating effector cells that eliminate infected and malignant cells. Following clearance, most effector cells terminally differentiate, while a subset of memory precursor effector cells survive contraction, giving rise to circulating memory T cells providing long-term protection. Some precursor cells infiltrate tissues, becoming tissue-resident memory T (TRM) cells. TRM cells persist in peripheral tissues, preventing reinfection and tumor growth. This project focuses on understanding the molecular networks controlling TRM tissue infiltration and long-term persistence with the final aim to optimize and improve adoptive cell transfer immunotherapy in solid cancers. Despite the success of chimeric antigen receptor (CAR) T cells in liquid tumors, their efficacy in solid tumors is hindered by immunosuppression, metabolic factors, and physical barriers, compromising functionality and tumor infiltration. Indeed, anti-tumor T cell responses resembling TRM correlate with improved prognosis.1 2 However, the molecular networks regulating adaptation to persistent antigen stimulation and tumor infiltration in CAR T cell therapy remain unknown. Thus, targeting solid tumors is still particularly challenging.MethodsTo study the infiltration of CAR T cells into solid tumors, we are conducting co-transductions of CD8 T cells using a CAR specific to the CD19 molecule (CAR19), along with genes encoding proteins of interest. The modified cells are then tested both in vitro and in vivo using tumor models, such as B16 mouse melanoma tumors that overexpress the CD19 molecule (B16.CD19).ResultsOur hypothesis is that providing tissue residency transcriptional programs to CAR T cells can enhance their ability to control solid tumors. As a proof of concept, we have simultaneously overexpressed the CAR and the transcription factor RUNX3, master regulator of tissue residency. This approach aims to promote tumor infiltration and T cell accumulation within the tumor. In vitro coculture experiments using the modified T cells and the B16.CD19 cell line have confirmed the specific killing activity of the co-transduced CAR T cells, regardless the overexpression of RUNX3. In vivo, tumor monitoring has revealed that RUNX3+CAR19+ CD8 T cells present superior control of tumor growth over time and higher accumulation within the tumor compared to RUNX3–CAR19+ cells. Notably, the RUNX3+CAR19+ T cells display increased expression of T-bet and CD44, suggesting a heightened effector potential, and of PD-1 and TCF-1, a phenotype associated with favorable prognosis in cancer patients.ConclusionsThese results highlight the importance of dissecting tissue-resident memory cells and their regulatory circuits to treat solid tumors.ReferencesJung IY, et al. Tissue-resident memory CAR T cells with stem-like characteristics display enhanced efficacy against solid and liquid tumors. Cell Rep Med, 2023:101053.Park SL, T Gebhardt, LK Mackay, Tissue-Resident Memory T Cells in Cancer Immunosurveillance. Trends Immunol, 2019;40(8):735–747.Ethics ApprovalAll animal studies were approved by the Institutional Animal Care and Use Committees of the University of California, San Diego (UCSD, protocol #S04105) and performed in accordance with UC guidelines.

During acute viral infections, clearance of the pathogen is followed by the contraction of the anti-viral T cell compartment. In contrast, T cell responses need to be maintained over a longer period of time during chronic viral infections in order to control viral replication and to avoid viral spreading. Much is known about inhibitory signals such as through PD-1 that limit T cell activity during chronic viral infection, but little is known about the stimulatory signals that allow maintenance of anti-viral T cells. Here, we show that the co-stimulatory molecule OX40 (CD134) is critically required in the context of persistent LCMV clone 13 infection. Anti-viral T cells express high levels of OX40 in the presence of their cognate antigen and T cells lacking the OX40 receptor fail to accumulate sufficiently. Moreover, the emergence of T cell dependent germinal center responses and LCMV-specific antibodies are severely impaired. Consequently, OX40-deficient mice fail to control LCMV clone 13 infection over time, highlighting the importance of this signaling pathway during persistent viral infection.

Data on therapy of COVID-19 in immunocompetent and immunosuppressed children are scarce. We aimed to explore management strategies of pediatric rheumatologists. All subscribers to international Pediatric Rheumatology Bulletin Board were invited to take part in an online survey on therapeutic approaches to COVID-19 in healthy children and children with autoimmune/inflammatory diseases (AID). Off-label therapies would be considered by 90.3% of the 93 participating respondents. In stable patients with COVID-19 on oxygen supply (stage I), use of remdesivir (48.3%), azithromycin (26.6%), oral corticosteroids (25.4%) and/or hydroxychloroquine (21.9%) would be recommended. In case of early signs of “cytokine storm” (stage II) or in critically ill patients (stage III) (a) anakinra (79.5% stage II; 83.6% stage III) or tocilizumab (58.0% and 87.0%, respectively); (b) corticosteroids (oral 67.2% stage II, intravenously 81.7% stage III); (c) intravenous immunoglobulins (both stages 56.5%); or (d) remdesivir (both stages 46.7%) were considered. In AID, > 94.2% of the respondents would not support a preventive adaptation of the immunomodulating therapy. In case of mild COVID-19, more than 50% of the respondents would continue pre-existing treatment with immunoglobulins (100%), hydroxychloroquine (94.2%), anakinra (79.2%) or canakinumab (72.5%), or tocilizumab (69.8%). Long-term corticosteroids would be reduced by 26.9% (< = 2 mg/kg/d) and 50.0% (> 2 mg/kg/day), respectively, with only 5.8% of respondents voting to discontinue the therapy. Conversely, more than 75% of respondents would refrain from administering cyclophosphamide and anti-CD20-antibodies. As evidence on management of pediatric COVID-19 is incomplete, continuous and critical expert opinion and knowledge exchange is helpful.

BackgroundIn cancer, CD8+ T cells have the power to target and kill tumor cells with precision, but often fail due to chronic activation from the immunosuppressive tumor microenvironment (TME). T cells that experience prolonged activation in the TME differentiate into a severely dysfunctional cell state known as exhaustion. In healthy tissues, T cells differentiate into tissue-resident memory cells (TRM) in response to infection, which remain lodged in tissues to provide protection from reinfection. When TRM-like cells are found in patient tumors, they are correlated with improved outcomes and responses to immunotherapy. Understanding how to manipulate T cell fates in an effort to prevent exhaustion and favor TRM-characteristics could benefit cancer immunotherapy.MethodsTo explore differences between these cell states, we screened the core TRM gene-expression signatures for genes downregulated as T cells undergo terminal exhaustion. Targets were then overexpressed in antigen-specific T cells and adoptively transferred into tumor-bearing mice for analysis.ResultsInterestingly, many genes related to protein regulation and processing were identified, including a novel gene called Neuralized E3 Ubiquitin Protein Ligase 3 (Neurl3). Neurl3's function is not well described, however, experimentally mutating the RING domain suggests Neurl3 transfers ubiquitin to target proteins for degradation. When Neurl3 was overexpressed in tumor-specific T cells, we found tumor infiltrating lymphocytes still upregulated inhibitory receptors PD1 and Tim3, but showed enhanced anti-tumor function. Neurl3-overexpressing T cells had increased accumulation in the TME, upregulated canonical TRM markers CD69 and CD103, produced more cytokines, controlled tumor growth, and increased mouse survival in B16 melanoma.ConclusionsThese results highlight the understudied field of negative regulation of T cell function by protein degradation in T cell differentiation fate and uncover a potential gene target for immunocellular therapies to favor functional T cell fates in cancer.Ethics ApprovalThe study was approved by UCSD's Institutional Animal Care and Use Committee, protocol number S04105.