Aberrant tolerogenic functions and proinflammatory skew of dendritic cells in STAT1 gain-of-function patients may contribute to autoimmunity and fungal susceptibility.

STAT1 gain-of-function (GOF) mutations underlie an inborn error of immunity hallmarked by chronic mucocutaneous candidiasis (CMC). Beyond the fungal susceptibility, attributed to Th17 failure, over half of the reported patients suffer from autoimmune manifestations, mechanism of which has not been explained yet. We hypothesized that the STAT1 mutations would affect dendritic cells' (DCs) properties and alter their inflammatory and tolerogenic functions. To test the hypothesis, we generated monocyte-derived DCs (moDCs) and tolerogenic DCs (tDCs). Functional and signaling studies, co-culture experiments and RNA sequencing demonstrated that STAT1 GOF DCs were profoundly altered in their phenotype and functions, characterized by loss of tolerogenic functions, proinflammatory skew and decreased capacity to induce Th17. Cytokine signaling, autophagy and metabolic processes were identified as the most prominently altered cellular processes. The results suggest that DCs are directly involved in STAT1 GOF-associated immune pathology, possibly contributing to both autoimmune manifestations and the failure of antifungal defense.

Th1/interferon-γ bias in 22q11.2 deletion syndrome is driven by memory T cells and exacerbated by IL-7.

The aim of this study was to investigate the impact of thymic dysplasia on the phenotypic and functional characteristics of T cells in patients with 22q11.2 deletion syndrome, including T-cell phenotype, transcriptional profile, cytokine production, as well as the possibility of utilizing IL-7 to recover their numbers and function. We found a strong bias towards Th1 response in pediatric and young adult 22q11.2DS patients, expansion of CXCR5+ follicular helper cells and CXCR3+CCR6- Th1 cells, increased production of cytokines IFN-γ, IL-10, IL-2, IL-21 and TNF-α. This Th1 skew was primarily driven by expanded terminally differentiated T cells. IL-7 further reduced naive T cells, increased cytokine production and caused an upregulation of exhaustion markers. Thus, Th1 bias in T cell populations persists from infancy into adolescence and is accompanied by accelerated maturation of T cells into memory stages. This phenotype is exacerbated by IL-7 which causes further decrease in naïve T cells in vitro.

Neutrophils in STAT1 Gain-Of-Function Have a Pro-inflammatory Signature Which Is Not Rescued by JAK Inhibition.

STAT1 gain-of-function (GOF) mutations cause an inborn error of immunity with diverse phenotype ranging from chronic mucocutaneous candidiasis (CMC) to various non-infectious manifestations, the most precarious of which are autoimmunity and vascular complications. The pathogenesis centers around Th17 failure but is far from being understood. We hypothesized that neutrophils, whose functions have not been explored in the context of STAT1 GOF CMC yet, might be involved in the associated immunodysregulatory and vascular pathology. In a cohort of ten patients, we demonstrate that STAT1 GOF human ex-vivo peripheral blood neutrophils are immature and highly activated; have strong propensity for degranulation, NETosis, and platelet-neutrophil aggregation; and display marked inflammatory bias. STAT1 GOF neutrophils exhibit increased basal STAT1 phosphorylation and expression of IFN stimulated genes, but contrary to other immune cells, STAT1 GOF neutrophils do not display hyperphosphorylation of STAT1 molecule upon stimulation with IFNs. The patient treatment with JAKinib ruxolitinib does not ameliorate the observed neutrophil aberrations. To our knowledge, this is the first work describing features of peripheral neutrophils in STAT1 GOF CMC. The presented data suggest that neutrophils may contribute to the immune pathophysiology of the STAT1 GOF CMC.

Mutual alteration of NOD2-associated Blau syndrome and IFNγR1 deficiency.

Blau syndrome (BS) is an auto-inflammatory granulomatous disease that possibly involves abnormal response to interferon gamma (IFNγ) due to exaggerated nucleotide-binding oligomerization domain containing 2 (NOD2) activity. Mendelian susceptibility to mycobacterial diseases (MSMD) is an infectious granulomatous disease that is caused by impaired production of or response to IFNγ. We report a mother and daughter who are both heterozygous for NOD2c.2264C˃T variant and dominant-negative IFNGR1818del4 mutation. The 17-year-old patient displayed an altered form of BS and milder form of MSMD, whereas the 44-year-old mother was completely asymptomatic. This experiment of nature supports the notion that IFNγ is an important driver of at least some BS manifestations and that elucidation of its involvement in the disease immunopathogenesis may identify novel therapeutic targets.

Monocytes contribute to DNA sensing through the TBK1 signaling pathway in type 1 diabetes patients.

BACKGROUND: The aberrant recognition of self-nucleic acids by the innate immune system contributes to the pathology of several autoimmune diseases. Although microbial DNA and, in certain instances, self-DNA that is released from damaged cells are primarily recognized by Toll-like receptor 9 (TLR9), recent evidence suggests that other cytosolic sequence-nonspecific DNA sensors contribute to DNA recognition. In this study, we focused on the sensing of microbial and host DNA in type 1 diabetes (T1D) patients. METHODS: Peripheral blood mononuclear cells (PBMCs) and monocytes from pediatric patients with T1D and from healthy donors were stimulated with microbial DNA (CpG) or with self-DNA (DNA contained within neutrophil extracellular traps, NETs). The production of cytokines was measured by flow cytometry and multiplex bead assays. The internalization of microbial DNA and its colocalization with STING was detected by image cytometry. Furthermore, the involvement of the TBK1 kinase was investigated by detecting its phosphorylation with phospho-flow cytometry or by using a TBK1 inhibition assay. RESULTS: We observed a prominent proinflammatory response in T1D PBMCs, especially pDCs and monocytes, to microbial DNA in comparison to that in controls. We further confirmed that monocytes could bind and internalize DNA and respond by releasing proinflammatory cytokines in a more pronounced manner in T1D patients than those in controls. Surprisingly, this cytokine production was not affected by TLR9 blockade, suggesting the involvement of intracellular receptors in DNA recognition. We further identified TBK1 and STING as two crucial molecules in the DNA-sensing pathway that were involved in CpG-DNA sensing by T1D cells. A similar DNA-sensing pathway that was dependent on intracellular DNA sensors and the STING-TBK1 interaction was employed in response to NETs, which were used to model self-DNA. CONCLUSIONS: Here, we show that there were significant differences in DNA sensing in T1D patients compared to that in controls. We demonstrate that monocytes from T1D patients are able to sense microbial- and self-DNA, leading to proinflammatory cytokine secretion through the adaptor protein STING and the TBK1 kinase.

Utility of Ruxolitinib in a Child with Chronic Mucocutaneous Candidiasis Caused by a Novel STAT1 Gain-of-Function Mutation.

PURPOSE: Signal transducer and activator of transcription 1 gain-of-function (STAT1 GOF) mutations are the most common cause of chronic mucocutaneous candidiasis (CMC). We aim to report the effect of oral ruxolitinib, the Janus kinase (JAK) family tyrosine kinase inhibitor, on clinical and immune status of a 12-year-old boy with severe CMC due to a novel STAT1 GOF mutation. METHODS: Clinical features and laboratory data were analyzed, particularly lymphocyte subsets, ex vivo IFNγ- and IFNα-induced STAT1, 3, 5 phosphorylation dynamics during the course of JAK1/2 inhibition therapy, and Th17-related, STAT1- and STAT3-inducible gene expression before and during the treatment. Sanger sequencing was used to detect the STAT1 mutation. Literature review of ruxolitinib in treatment of CMC is appended. RESULTS: A novel STAT1 GOF mutation (c.617T > C; p.L206P), detected in a child with recalcitrant CMC, was shown to be reversible in vitro with ruxolitinib. Major clinical improvement was achieved after 8 weeks of ruxolitinib treatment, while sustained suppression of IFNγ- and IFNα-induced phosphorylation of STAT1, STAT3, and STAT5, as well as increased STAT3-inducible and Th17-related gene expression, was demonstrated ex vivo. Clinical relapse and spike of all monitored phosphorylated STAT activity was registered shortly after unplanned withdrawal, decreasing again after ruxolitinib reintroduction. No increase of peripheral CD4+ IL17+ T cells was detected after 4 months of therapy. No adverse effects were noted. CONCLUSION: JAK1/2 inhibition with ruxolitinib represents a viable option for treatment of refractory CMC, if HSCT is not considered. However, long-term administration is necessary, as the effect is not sustained after treatment discontinuation.

Elevated Biomarkers of NETosis in the Serum of Pediatric Patients With Type 1 Diabetes and Their First-Degree Relatives.

Type 1 diabetes (T1D) is an autoimmune disorder with unambiguous involvement of both innate and adaptive immune mechanisms in the destruction of pancreatic beta cells. Recent evidence demonstrated that neutrophils infiltrate the pancreas prior to disease onset and therein extrude neutrophil extracellular traps (NETs), web-like structures of DNA and nuclear proteins with a strong pro-inflammatory biologic activity. Our previous work showed that T1D NETs activate dendritic cells, which consequently induce IFNγ-producing Th1 lymphocytes. The aim of this study was to assess direct ex vivo biomarkers of NETosis in the serum of recent onset and long-term pediatric T1D patients, their first-degree relatives and healthy controls. To this end we evaluated serum levels of myeloperoxidase (MPO), neutrophil elastase (NE), proteinase 3 (PR3), protein arginine deiminase 4 (PAD4), LL37 and cell-free DNA-histone complexes in sex- and age-matched cohorts of T1D first-degree relatives, recent-onset T1D patients, and in patients 12 months after clinical manifestation of the disease. Our data shows that disease onset is accompanied by peripheral neutrophilia and significant elevation of MPO, NE, PR3, PAD4 and cell-free DNA-histone complexes. Most biomarkers subsequently decrease but do not always normalize in long-term patients. First-degree relatives displayed an intermediate phenotype, except for remarkably high levels of LL37. Together, this report provides evidence for the presence of ongoing NETosis in pediatric patients with T1D at time of clinical manifestation of the disease, which partly subsides in subsequent years.

Highly active antimycobacterial derivatives of benzoxazine.

New 3-(4-alkylphenyl)-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones and 3-(4-alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-dithiones were synthesized. The compounds were tested for in vitro antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium avium and two strains of Mycobacterium kansasii. The antimycobacterial activity increased with the replacement of the carbonyl group by the thiocarbonyl group in the starting 3-(4-alkylphenyl)-2H-1,3-benzoxazine-2,4(3H)-diones. The most active derivatives were more active than isonicotinhydrazide (INH). Free-Wilson analysis was also carried out and the activity contribution was examined.

Neutrophil Extracellular Trap Induced Dendritic Cell Activation Leads to Th1 Polarization in Type 1 Diabetes.

Neutrophils releasing neutrophil extracellular traps (NETs) infiltrate the pancreas prior to type 1 diabetes (T1D) onset; however, the precise nature of their contribution to disease remains poorly defined. To examine how NETs affect immune functions in T1D, we investigated NET composition and their effect on dendritic cells (DCs) and T lymphocytes in T1D children. We showed that T1D patient NET composition differs substantially from that of healthy donors and that the presence of T1D-NETs in a mixed peripheral blood mononuclear cell culture caused a strong shift toward IFNγ-producing T lymphocytes, mediated through activation of innate immunity cells in T1D samples. Importantly, in a monocyte-derived DC (moDC) culture, NETs induced cytokine production, phenotypic change and IFNγ-producing T cells only in samples from T1D patients but not in those from healthy donors. RNA-seq analysis revealed that T1D-NETs presence causes TGFß downregulation and IFNα upregulation and creates pro-T1D signature in healthy moDCs.

Novel XIAP mutation causing enhanced spontaneous apoptosis and disturbed NOD2 signalling in a patient with atypical adult-onset Crohn's disease.

X-linked inhibitor of apoptosis (XIAP) is the most potent human inhibitor of apoptosis, and is also involved in NOD2-dependent NFκB and MAPK signalling cascade activation. The absence or defective function of XIAP leads to the development of a rare and severe primary immunodeficiency known as X-linked lymphoproliferative syndrome type 2 (XLP-2), which is characterized by a triad of clinical manifestations, including a high incidence of haemophagocytic lymphohistiocytosis (HLH), lymphoproliferation and inflammatory bowel disease (IBD), usually with very early onset. Here, we present a novel XIAP mutation identified in a patient with atypical adult-onset IBD complicated by relapsing HLH, splenomegaly and sarcoid-like disease. The c.266delA mutation in the XIAP gene creates a premature stop codon, and causes a severe reduction in XIAP protein expression. The mutation is also associated with impaired spontaneous and staurosporine- and PMA-induced apoptosis accompanied by significantly increased expression of pro-apoptotic genes. We also confirmed the negative impact of this particular XIAP mutation on NOD2-dependent NFκB and MAPK activation, while NOD2-independent activation was found to be unaffected. Moreover, we assume that the mutation has an impact on the overproduction of IL-12 and IFNγ, the shift towards the Th1 immune response and increased numbers of central memory and effector memory CD4+ and CD8+ T cells. All these changes contribute to immune dysregulation and the clinical manifestation of XLP-2.

Complex Immunometabolic Profiling Reveals the Activation of Cellular Immunity and Biliary Lesions in Patients with Severe COVID-19.

This study aimed to assess the key laboratory features displayed by coronavirus disease 2019 (COVID-19) inpatients that are associated with mild, moderate, severe, and fatal courses of the disease, and through a longitudinal follow-up, to understand the dynamics of the COVID-19 pathophysiology. All severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive patients admitted to the University Hospital in Motol between March and June 2020 were included in this study. A severe course of COVID-19 was associated with an elevation of proinflammatory markers; an efflux of immature granulocytes into peripheral blood; the activation of CD8 T cells, which infiltrated the lungs; transient liver disease. In particular, the elevation of serum gamma-glutamyl transferase (GGT) and histological signs of cholestasis were highly specific for patients with a severe form of the disease. In contrast, patients with a fatal course of COVID-19 failed to upregulate markers of inflammation, showed discoordination of the immune response, and progressed toward acute kidney failure. COVID-19 is a disease with a multi-organ affinity that is characterized by the activation of innate and cellular adaptive immunity. Biliary lesions with an elevation of GGT and the organ infiltration of interleukin 6 (IL-6)-producing cells are the defining characteristics for patients with the fulminant disease.

Disharmonic Inflammatory Signatures in COVID-19: Augmented Neutrophils' but Impaired Monocytes' and Dendritic Cells' Responsiveness.

COVID-19, caused by SARS-CoV-2 virus, emerged as a pandemic disease posing a severe threat to global health. To date, sporadic studies have demonstrated that innate immune mechanisms, specifically neutrophilia, NETosis, and neutrophil-associated cytokine responses, are involved in COVID-19 pathogenesis; however, our understanding of the exact nature of this aspect of host-pathogen interaction is limited. Here, we present a detailed dissection of the features and functional profiles of neutrophils, dendritic cells, and monocytes in COVID-19. We portray the crucial role of neutrophils as drivers of hyperinflammation associated with COVID-19 disease via the shift towards their immature forms, enhanced degranulation, cytokine production, and augmented interferon responses. We demonstrate the impaired functionality of COVID-19 dendritic cells and monocytes, particularly their low expression of maturation markers, increased PD-L1 levels, and their inability to upregulate phenotype upon stimulation. In summary, our work highlights important data that prompt further research, as therapeutic targeting of neutrophils and their associated products may hold the potential to reduce the severity of COVID-19.

Data on microbial DNA-induced IL-1ß production in monocytes of type 1 diabetes patients.

Inflammasomes are large protein complexes involved in the maturation of IL-1ß, a cytokine associated with the pathophysiology of type 1 diabetes (T1D). The data presented in this article focused on the role of inflammasomes in DNA recognition in T1D patients. This data extend knowledge on DNA sensing in T1D patients and relate to our research paper "Monocytes contribute to DNA sensing through the TBK1 signaling pathway in type 1 diabetes patients" Zentsova et al., 2009. To examine inflammasome involvement, we blocked the known mechanism of inflammasome activation - potassium efflux via various approaches: 1) high concentration of KCl; 2) Glybenclamide, which selectively blocks the ATP sensitive K+ channel; 3) KN-62, an inhibitor of P2X7 receptor, which activates K+ channel after ATP binding. Moreover, we used an inhibitor which blocks Nod-like receptor family containing pyrin domain 3 (NLRP3) inflammasome. In T1D patients, we show that secretion of cytokines IL-1ß, TNFα, IL-6 and IFNα after microbial DNA stimulation is promoted by potassium efflux and is not dependent on P2X7 receptor signaling. Surprisingly, the microbial DNA induced IL-1ß release was independent of NLRP3.

Generation of T cell effectors using tumor cell-loaded dendritic cells for adoptive T cell therapy.

Adoptive T cell transfer has been shown to be an effective method used to boost tumor-specific immune responses in several types of malignancies. In this study, we set out to optimize the ACT protocol for the experimental treatment of prostate cancer. The protocol includes a pre-stimulation step whereby T cells were primed with autologous dendritic cells loaded with the high hydrostatic pressure-treated prostate cancer cell line, LNCaP. Primed T cells were further expanded in vitro with anti-CD3/CD28 Dynabeads in the WAVE bioreactor 2/10 system and tested for cytotoxicity. Our data indicates that the combination of pre-stimulation and expansion steps resulted in the induction and enrichment of tumor-responsive CD4+ and CD8+ T cells at clinically relevant numbers. The majority of both CD4+ and CD8+ IFN-γ producing cells were CD62L, CCR7 and CD57 negative but CD28 and CD27 positive, indicating an early antigen experienced phenotype in non-terminal differentiation phase. Expanded T cells showed significantly greater cytotoxicity against LNCaP cells compared to the control SKOV-3, an ovarian cancer line. In summary, our results suggest that the ACT approach together with LNCaP-loaded dendritic cells provides a viable way to generate prostate cancer reactive T cell effectors that are capable of mounting efficient and targeted antitumor responses and can be thus considered for further testing in a clinical setting.

Phase I/II clinical trial of dendritic-cell based immunotherapy (DCVAC/PCa) combined with chemotherapy in patients with metastatic, castration-resistant prostate cancer.

PURPOSE: We conducted an open-label, single-arm Phase I/II clinical trial in metastatic CRPC (mCRPC) patients eligible for docetaxel combined with treatment with autologous mature dendritic cells (DCs) pulsed with killed LNCaP prostate cancer cells (DCVAC/PCa). The primary and secondary endpoints were safety and immune responses, respectively. Overall survival (OS), followed as a part of the safety evaluation, was compared to the predicted OS according to the Halabi and MSKCC nomograms. EXPERIMENTAL DESIGN: Twenty-five patients with progressive mCRPC were enrolled. Treatment comprised of initial 7 days administration of metronomic cyclophosphamide 50 mg p.o. DCVAC/PCa treatment consisted of a median twelve doses of 1 × 107 dendritic cells per dose injected s.c. (Aldara creme was applied at the site of injection) during a one-year period. The initial 2 doses of DCVAC/PCa were administered at a 2-week interval, followed by the administration of docetaxel (75 mg/m2) and prednisone (5 mg twice daily) given every 3 weeks until toxicity or intolerance was observed. The DCVAC/PCa was then injected every 6 weeks up to the maximum number of doses manufactured from one leukapheresis. RESULTS: No serious DCVAC/PCa-related adverse events have been reported. The median OS was 19 months, whereas the predicted median OS was 11.8 months with the Halabi nomogram and 13 months with the MSKCC nomogram. Kaplan-Meier analyses showed that patients had a lower risk of death compared with both MSKCC (Hazard Ratio 0.26, 95% CI: 0.13-0.51) and Halabi (Hazard Ratio 0.33, 95% CI: 0.17-0.63) predictions. We observed a significant decrease in Tregs in the peripheral blood. The long-term administration of DCVAC/PCa led to the induction and maintenance of PSA specific T cells. We did not identify any immunological parameter that significantly correlated with better OS. CONCLUSIONS: In patients with mCRPC, the combined chemoimmunotherapy with DCVAC/PCa and docetaxel was safe and resulted in longer than expected survival. Concomitant chemotherapy did not preclude the induction of specific anti-tumor cytotoxic T cells.