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Patients with inborn errors of the alternative NF-κB pathway have low thymic AIRE expression, leading to the development of auto-Abs neutralizing type I IFNs, and severe viral diseases.

Summary: We characterized Mycobacterium bovis BCG isolates found in lung and brain samples from a previously vaccinated patient with IFNγR1 deficiency. The isolates collected displayed distinct genomic and phenotypic features consistent with host adaptation and associated changes in antibiotic susceptibility and virulence traits. Background: We report a case of a patient with partial recessive IFNγR1 deficiency who developed disseminated BCG infection after neonatal vaccination (BCG-vaccine). Distinct M. bovis BCG-vaccine derived clinical strains were recovered from the patient’s lungs and brain. Methods: BCG strains were phenotypically (growth, antibiotic susceptibility, lipid) and genetically (whole genome sequencing) characterized. Mycobacteria cell infection models were used to assess apoptosis, necrosis, cytokine release, autophagy, and JAK-STAT signaling. Results: Clinical isolates BCG-brain and BCG-lung showed distinct Rv0667 rpoB mutations conferring high- and low-level rifampin resistance; the latter displayed clofazimine resistance through Rv0678 gene (MarR-like transcriptional regulator) mutations. BCG-brain and BCG-lung showed mutations in fadA2, fadE5, and mymA operon genes, respectively. Lipid profiles revealed reduced levels of PDIM in BCG-brain and BCG-lung and increased TAGs and Mycolic acid components in BCG-lung, compared to parent BCG-vaccine. In vitro infected cells showed that the BCG-lung induced a higher cytokine release, necrosis, and cell-associated bacterial load effect when compared to BCG-brain; conversely, both strains inhibited apoptosis and altered JAK-STAT signaling. Conclusions: During a chronic-disseminated BCG infection, BCG strains can evolve independently at different sites likely due to particular microenvironment features leading to differential antibiotic resistance, virulence traits resulting in dissimilar responses in different host tissues.

Mycobacterium genavense is a challenging opportunistic pathogen to diagnose and manage in patients with human immunodeficiency virus (HIV). Persistent immunosuppression or protracted immune reconstitution inflammatory syndrome can lead to complicated clinical courses. We describe 3 cases of M. genavense in patients with HIV representing the spectrum between disease burden and strength of immune response.

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).

Dedicator-of-cytokinesis 8 (DOCK8) deficiency, a primary immunodeficiency disease, can be reversed by allogeneic hematopoietic stem cell transplantation (HSCT); however, there are few reports describing the use of alternative donor sources for HSCT in DOCK8 deficiency. We describe HSCT for patients with DOCK8 deficiency who lack a matched related or unrelated donor using bone marrow from haploidentical related donors and post-transplantation cyclophosphamide (PT/Cy) for graft-versus-host disease (GVHD) prophylaxis. Seven patients with DOCK8 deficiency (median age, 20 years; range, 7 to 25 years) received a haploidentical related donor HSCT. The conditioning regimen included 2 days of low-dose cyclophosphamide, 5 days of fludarabine, 3 days of busulfan, and 200 cGy total body irradiation. GVHD prophylaxis consisted of PT/Cy 50 mg/kg/day on days +3 and +4 and tacrolimus and mycophenolate mofetil starting at day +5. The median times to neutrophil and platelet engraftment were 15 and 19 days, respectively. All patients attained >90% donor engraftment by day +30. Four subjects developed acute GVHD (1 with maximum grade 3). No patient developed chronic GVHD. With a median follow-up time of 20.6 months (range, 9.5 to 31.7 months), 6 of 7 patients are alive and disease free. Haploidentical related donor HSCT with PT/Cy represents an effective therapeutic approach for patients with DOCK8 deficiency who lack a matched related or unrelated donor. Published by Elsevier Inc. on behalf of the American Society for Blood and Marrow Transplantation.

Lyons et al. show that STAT3 negatively regulates TGF-β signaling via ERBIN and that cell-intrinsic deregulation of TGF-β pathway activation promotes the IL-4/IL-4Rα/GATA3 axis to support atopic phenotypes in humans., Nonimmunological connective tissue phenotypes in humans are common among some congenital and acquired allergic diseases. Several of these congenital disorders have been associated with either increased TGF-β activity or impaired STAT3 activation, suggesting that these pathways might intersect and that their disruption may contribute to atopy. In this study, we show that STAT3 negatively regulates TGF-β signaling via ERBB2-interacting protein (ERBIN), a SMAD anchor for receptor activation and SMAD2/3 binding protein. Individuals with dominant-negative STAT3 mutations (STAT3mut) or a loss-of-function mutation in ERBB2IP (ERBB2IPmut) have evidence of deregulated TGF-β signaling with increased regulatory T cells and total FOXP3 expression. These naturally occurring mutations, recapitulated in vitro, impair STAT3–ERBIN–SMAD2/3 complex formation and fail to constrain nuclear pSMAD2/3 in response to TGF-β. In turn, cell-intrinsic deregulation of TGF-β signaling is associated with increased functional IL-4Rα expression on naive lymphocytes and can induce expression and activation of the IL-4/IL-4Rα/GATA3 axis in vitro. These findings link increased TGF-β pathway activation in ERBB2IPmut and STAT3mut patient lymphocytes with increased T helper type 2 cytokine expression and elevated IgE.

Background Sjögren’s syndrome (SS) is an autoimmune disease characterized by immune attack on the salivary and lacrimal glands. Given the known cytokine activation and type I interferon gene expression signature found in SS, we hypothesized that anticytokine autoantibodies might be detectable by Luciferase immunoprecipitation systems in some SS patients and correlate with clinical symptoms. Results Luciferase immunoprecipitation systems was used to screen for serum anti-cytokine autoantibodies in 57 primary SS patients and 25 healthy volunteers. Autoantibodies were detected against GMCSF, interferon-γ, -α and, -ω in one, two, two and six patients with SS, respectively. None of the healthy volunteers showed anticytokine autoantibodies and none of the SS or control subjects showed autoantibodies against interferon-λ. One 51-year old female SS subject with the highest anti-interferon-α and -ω autoantibody levels had stable autoantibody levels over the course of a year. In vitro functional testing of serum autoantibodies from this subject demonstrated partially neutralizing activity for interferon-α signaling. Clinical information on this individual revealed a low focus score and high levels of unstimulated salivary flow, suggesting the possibility that interferon-α autoantibody neutralizing activity may have contributed to the milder sicca symptoms. Conclusion Overall, these findings demonstrate that a subset of SS patients (16%) harbor autoantibodies against GMCSF, interferon-γ, interferon-ω, and interferon-α. These data support the observation that high levels of interferon-α autoantibodies may attenuate disease symptoms in SS.

Treatment of PML with Pembrolizumab A patient with variable immunodeficiency and progressive multifocal leukoencephalopathy was treated with five pembrolizumab infusions. The CSF JC viral load went...

We present a 17-year old girl with DOCK-8 deficiency, severe untreated oral HSV-1 infection and associated aggressive periodontitis. DOCK-8 deficiency is a primary immunodeficiency, caused by biallelicloss-of-function mutations in the DOCK8 gene, often leading to severe viral and fungal mucocutaneous infections. Nevertheless, to date DOCK8 has not been associated with severe periodontitis and inflammatory bone loss around teeth. Understanding whether DOCK8 deficiency or severe HSV-1 infection underlies susceptibility to periodontitis is central to this case and may provide insights into susceptibility factors for periodontitis in the general population. Our clinical and microbiological data suggest that severe HSV-1 infection is the driver of periodontal inflammation in this case.

Monozygotic twins with GATA2 deficiency: same haploidentical-related donor, different severity of GvHD

Contains fulltext : 118131.pdf (Publisher’s version ) (Closed access) Invasive aspergillosis is a major threat to patients with chronic granulomatous disease (CGD). Fungal pathogenesis is the result of a diminished antifungal capacity and dysregulated inflammation. A deficient NADPH-oxidase complex results in defective phagolysosomal alkalization. To investigate the contribution of defective pH regulation in phagocytes among patients with CGD during fungal pathogenesis, we evaluated the effect of the acidotropic, antimalarial drug chloroquine (CQ) on the antifungal capacity of polymorphonuclear cells (PMNs) and on the inflammatory response of peripheral blood mononuclear cells (PBMCs). Chloroquine exerted a direct pH-dependent antifungal effect on Aspergillus fumigatus and Aspergillus nidulans; it increased the antifungal activity of PMNs from patients with CGD at a significantly lower concentration, compared with the concentration for PMNs from healthy individuals; and decreased the hyperinflammatory state of PBMCs from patients with CGD, as observed by decreased tumor necrosis factor alpha and interleukin 1beta release. Chloroquine targets both limbs of fungal pathogenesis and might be of great value in the clearance of invasive aspergillosis in patients with CGD.

Heterozygous GATA2 mutation is associated with immunodeficiency, lymphedema, and myelodysplastic syndrome. Disease presentation is variable, often coinciding with loss of circulating dendritic cells, monocytes, B cells, and natural killer (NK) cells. Nonetheless, in a proportion of patients carrying GATA2 mutation, NK cells persist. We found that peripheral blood NK cells in symptomatic patients uniformly lacked expression of the transcription factor promyelocytic leukemia zinc finger (PLZF), as well as expression of intracellular signaling proteins FceRγ, spleen tyrosine kinase (SYK), and EWS/FLI1-Activated Transcript 2 (EAT-2) in a variegated manner. Moreover, consistent with an adaptive identity, NK cells from patients with GATA2 mutation displayed altered expression of cytotoxic granule constituents and produced interferon-γ upon Fc-receptor engagement but not following combined interleukin-12 (IL-12) and IL-18 stimulation. Canonical, PLZF-expressing NK cells were retained in asymptomatic carriers of GATA2 mutation. Developmentally, GATA-binding protein-2 (GATA-2) was expressed in hematopoietic stem cells, but not in NK-cell progenitors, CD3-CD56bright, canonical, or adaptive CD3-CD56dim NK cells. Peripheral blood NK cells from individuals with GATA2 mutation proliferated normally in vitro, whereas lineage-negative progenitors displayed impaired NK-cell differentiation. In summary, adaptive NK cells can persist in patients with GATA2 mutation, even after NK-cell progenitors expire. Moreover, our data suggest that adaptive NK cells are more long-lived than canonical, immunoregulatory NK cells.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency resulting from defects in the multienzyme complex NADPH-oxidase (phagozyte oxidase, phox), which normally produces microbicidal reactive oxygen metabolites (ROM). The reason for our patient's CGD was unusual, as revealed by the following in vitro findings in neutrophils and EBV-transformed B-cells: lack of flavocytochrome b(558) expression, restoration of significant ROM production after transduction with gp91-phox cDNA by a retrovirus vector, an 879G-->A, Trp289-->Stop mutation in one X chromosomal gp91-phox allele, a one-sided paternal X chromosome inactivation, as shown by a lyonization assay at the HUMARA locus, and the result of a dihydrorhodamine 123 flow cytometry assay revealing consistently that 1 in 2500 neutrophils produced ROM at normal levels. Our conclusion: A presumed autosomal form of CGD has been excluded. Instead, a spontaneous mutation in gp91-phox coinciding with an extreme X chromosome inactivation ratio resulted in X-linked CGD in this young woman.

Familial Mediterranean fever (FMF) has traditionally been considered an autosomal-recessive disease; however, it has been observed that a substantial number of patients with clinical FMF possess only 1 demonstrable MEFV mutation. The purpose of this study was to perform an extensive search for a second MEFV mutation in 46 patients diagnosed clinically as having FMF and carrying only 1 high-penetrance FMF mutation.MEFV and other candidate genes were sequenced by standard capillary electrophoresis. In 10 patients, the entire 15-kb MEFV genomic region was resequenced using hybridization-based chip technology. MEFV gene expression levels were determined by quantitative reverse transcription-polymerase chain reaction. Pyrin protein levels were examined by Western blotting.A second MEFV mutation was not identified in any of the patients who were screened. Haplotype analysis did not identify a common haplotype that might be associated with the transmission of a second FMF allele. Western blots did not demonstrate a significant difference in pyrin levels between patients with a single mutation and those with a double mutation; however, FMF patients of both types showed higher protein expression as compared with controls and with non-FMF patients with active inflammation. Screening of genes encoding pyrin-interacting proteins identified rare mutations in a small number of patients, suggesting the possibility of digenic inheritance.Our data underscore the existence of a significant subset of FMF patients who are carriers of only 1 MEFV mutation and demonstrate that complete MEFV sequencing is not likely to yield a second mutation. Screening for the set of the most common mutations and detection of a single mutation appears to be sufficient in the presence of clinical symptoms for the diagnosis of FMF and the initiation of a trial of colchicine.

were transplanted into recipients. 4 weeks post-transplantation, we treated mice with either 0, 0.5 or 2Gy radiation. Mice were monitored for aberrations in any of the hematopoietic lineages in both peripheral blood (PB) and bone marrow (BM) at frequent intervals. Mice developing t-MDS/AML like disease were sacrificed and ligation-mediated PCR (LM-PCR) was performed to identify the integration sites. Results: Ranging from 23-59 weeks post-radiation, 70% of the animals treated with 0.5Gy developed a tMDS/AML-like disease while interestingly only 17% of the 2Gy mice developed tMDS/ AML-like disease. None of the control animals developed tMDS/ AML-like disease, confirming specificity for t-MDS of our experimental setup. BM of mice with tMDS/AML-like disease showed a high level of donor chimerism and mice exhibited anemia, thrombocytopenia, splenomegaly and myeloid blasts in BM at different time points. In LM-PCR, we observed unique band patterns for each of the t-MDS/AML mice, indicating that the clones driving the disease were probably unique. Further gene expression analysis was performed on the genes located around the integration site to identify individual genes that are altered in these mice. Among others, C1q and tumor necrosis factor related protein 6 (c1qtnf6/ CTRP6), a fatty acid oxidation regulatory gene, Neuroblastoma ras oncogene (N-ras), a known oncogene in various cancers and miR10a, a microRNA regulating cell survival through Twist1 gene expression in myeloid cells, are the genes with altered expression in tMDS/AML mice. Thus generated presumptive t-MDS predisposing gene list further needs to be validated in vivo. Future directions: We anticipate a detailed validation of these genes in mice will facilitate the identification of genes that predispose to t-MDS/AML. We believe this is a unique mouse model mimicking a radiation mediated tMDS clinical scenario.

A girl with relapsing cervical lymphadenopathy due to Mycobacterium avium subsequently developed abdominal adenopathy and intestinal inflammation. 1 known (c.1623_1624delGCinsTT) and 1 novel mutation (c.65_68delCTGC of exon2) of the Interleukin-12 Receptor-beta1 (IL-12Rbeta1) gene was detected. Unlike reports of a more favorable outcome in these patients, our patient died of severe intestinal involvement.

Introduction DOCK8 mutations are responsible for a rare autosomal recessive immunodeficiency syndrome associated with severe cutaneous viral infections, elevated IgE levels, environmental allergies, autoimmunity, and malignancy. DOCK8 activates CDC42, which is important for cell signalling and actin reorganisation. Natural killer cells play a vital role in tumour surveillance and defence against virally infected cells. NK cell function relies on the accumulation of actin at the NK cell immunologic synapse formed with target cells. Although abnormalities in T and B cell function have been described in DOCK8-deficient patients, the role of NK cells in this disease is poorly understood. Objectives/Aims Given the susceptibility to severe cutaneous viral infection and malignancy, we hypothesised there was a substantive defect in NK cell function in patients with DOCK8 deficiency. Methods 10 patients with genetically confirmed DOCK8 deficiency as well as NK cell lines with stably reduced DOCK8 expression were evaluated experimentally using in vitro NK cell cytotoxicity, F-actin content, and confocal immunofluorescence microscopy assays. Results DOCK8-deficient patients and cell lines all had decreased NK cell cytotoxicity and function could not be restored after IL-2 stimulation. Importantly, DOCK8 deficiency did not affect NK cell F-actin content, but impaired F-actin accumulation at the lytic immunological synapse. Conclusions DOCK8 deficiency results in severely deficient NK cell function owing to an inability to form a mature lytic immunological synapse via focal F-actin accumulation. This defect may underlie important and previously perplexing attributes of the DOCK8 deficiency clinical syndrome including the unusual susceptibility to viral infection.

within 1 Mb of the TYK2 gene. Four of the five families had at least one unaffected child, and in three of these four, the genotype data were consistentwithgeneticlinkagetotheTYK2locus on chromosome 19. We subsequently performed sequence analysis on the genomic DNA of one patient from each of these five families, by using the primers for human TYK2 kindly provided by Minegishi et al. (2006). We did not find any mutation in the coding regions of exons and the adjacentintronicsequencesofTYK2.However,wedidnotanalyzethefullintronic regions and the promoter of the gene. We conclude that Tyk2 deficiency is most likely not a common cause of the AR-HIES and suggest that human Tyk2 deficiency is a distinct disease entity thatisgeneticallyandclinicallydifferent from the previously published patients with AR-HIES. This hypothesis is supported by atypical features in the clinical history of the patient described by Minegishi et al. (2006) ,s uch as a BCG lymphadenitis and a non-Typhi Salmonella infection. The susceptibility to these certain viral and intracellular bacterial infections may imply that human TYK2 deficiency is clinically and pathophysiologically closely related to patients with particular defects in the interleukin-12-interferon axis as described by Casanova and Abel (2004). Therefore, it might be rewarding to analyze TYK2 further in these cohorts in addition to patients with AR-HIES.

Rationale Hyper-IgE syndrome (HIES, Job syndrome) is characterized by a triad of elevated IgE, skin abscesses and cyst-forming pneumonia. Non-immunological characteristics of this syndrome (skeletal anomalies, scoliosis, retention of primary teeth, osteoporosis, pathologic fractures, characteristic facies and hyperextensibility) have been described in detail only recently. Some of these characteristics as well as the severe infectious complications may not be apparent until late childhood or adolescence. Our objective was to study the association of HIES with skeletal anomalies that are detectable in the early years. Methods We reviewed our data from 159 subjects in 49 families evaluated for possible HIES at the National Institutes of Health. Congenital skeletal anomalies were detected by radiological assessment of the skull, spine and long bones. Results HIES was diagnosed in 52 patients. Ten of these patients had not had either skin abscesses or a history of pneumonias by the time of diagnosis, but presented with the other components of this syndrome and/or a fully affected first degree relative. Seventeen of the 52 HIES patients (32%) had at least one congenital skeletal anomaly, in contrast to 5 of 107 (4.7%) subjects in whom HIES was not confirmed (p=0.002). Some of the observed skeletal anomalies were: craniosynostosis, hemivertebrae, short limbs, spina bifida, bifid rib and Blount's disease. Conclusions HIES patients have an increased incidence of congenital skeletal anomalies. Physicians should look for skeletal anomalies when considering HIES, especially in patients too young to have enough manifestations of HIES to make a definitive diagnosis.