IKAROS-how many feathers have you lost: mild and severe phenotypes in IKZF1 deficiency.

Heterozygous germline variants in human IKZF1 encoding for IKAROS define an inborn error of immunity with immunodeficiency, immune dysregulation and risk of malignancy with a broad phenotypic spectrum. Growing evidence of underlying pathophysiological genotype-phenotype correlations helps to improve our understanding of IKAROS-associated diseases. We describe 6 patients from 4 kindreds with two novel IKZF1 variants leading to haploinsufficiency from 3 centers in Germany. We also provide an overview of first symptoms to a final diagnosis including data from the literature.

The immunopathological landscape of human pre-TCRα deficiency: From rare to common variants.

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-α T cell receptor (pre-TCRα) expression. Low circulating naive αß T cell counts at birth persisted over time, with normal memory αß and high γδ T cell counts. Their TCRα repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue αß 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 αß T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αß T cells, autoimmune conditions were more frequent in these patients compared with the general population.

Screening Newborns for Low T Cell Receptor Excision Circles (TRECs) Fails to Detect Immunodeficiency, Centromeric Instability, and Facial Anomalies Syndrome.

BACKGROUND: Assessment of T-cell receptor excision circles (TRECs) in dried blood spots of newborns allows the detection of severe combined immunodeficiency (SCID) (T cells <300/µL at birth) with a presumed sensitivity of 100%. TREC screening also identifies patients with selected combined immunodeficiency (CID) (T cells >300/µL, yet <1500/µL at birth). Nevertheless, relevant CIDs that would benefit from early recognition and curative treatment pass undetected. OBJECTIVE: We hypothesized that TREC screening at birth cannot identify CIDs that develop with age. METHODS: We analyzed the number of TRECs in dried blood spots in archived Guthrie cards of 22 children who had been born in the Berlin-Brandenburg area between January 2006 and November 2018 and who had undergone hematopoietic stem-cell transplantation (HSCT) for inborn errors of immunity. RESULTS: All patients with SCID would have been identified by TREC screening, but only 4 of 6 with CID. One of these patients had immunodeficiency, centromeric instability, and facial anomalies syndrome type 2 (ICF2). Two of 3 patients with ICF whom we have been following up at our institution had TREC numbers above the cutoff value suggestive of SCID at birth. Yet all patients with ICF had a severe clinical course that would have justified earlier HSCT. CONCLUSIONS: In ICF, naïve T cells may be present at birth, yet they decline with age. Therefore, TREC screening cannot identify these patients. Early recognition is nevertheless crucial, as patients with ICF benefit from HSCT early in life.

In vitro and in vivo evidence that the switch from calcineurin to mTOR inhibitors may be a strategy for immunosuppression in Epstein-Barr virus-associated post-transplant lymphoproliferative disorder.

Post-transplant lymphoproliferative disorder is a life-threatening complication of immunosuppression following transplantation mediated by failure of T cells to control Epstein-Barr virus (EBV)-infected and transformed B cells. Typically, a modification or reduction of immunosuppression is recommended, but insufficiently defined thus far. In order to help delineate this, we characterized EBV-antigen-specific T cells and lymphoblastoid cell lines from healthy donors and in patients with a kidney transplant in the absence or presence of the standard immunosuppressants tacrolimus, cyclosporin A, prednisolone, rapamycin, and mycophenolic acid. Phenotypes of lymphoblastoid cell-lines and T cells, T cell-receptor-repertoire diversity, and T-cell reactivity upon co-culture with autologous lymphoblastoid cell lines were analyzed. Rapamycin and mycophenolic acid inhibited lymphoblastoid cell-line proliferation. T cells treated with prednisolone and rapamycin showed nearly normal cytokine production. Proliferation and the viability of T cells were decreased by mycophenolic acid, while tacrolimus and cyclosporin A were strong suppressors of T-cell function including their killing activity. Overall, our study provides a basis for the clinical decision for the modification and reduction of immunosuppression and adds information to the complex balance of maintaining anti-viral immunity while preventing acute rejection. Thus, an immunosuppressive regime based on mTOR inhibition and reduced or withdrawn calcineurin inhibitors could be a promising strategy for patients with increased risk of or manifested EBV-associated post-transplant lymphoproliferative disorder.

Robust hepatitis B vaccine-reactive T cell responses in failed humoral immunity.

While virus-specific antibodies are broadly recognized as correlates of protection, virus-specific T cells are important for direct clearance of infected cells. Failure to generate hepatitis B virus (HBV)-specific antibodies is well-known in patients with end-stage renal disease. However, whether and to what extent HBV-specific cellular immunity is altered in this population and how it influences humoral immunity is not clear. To address it, we analyzed HBV-reactive T cells and antibodies in hemodialysis patients post vaccination. 29 hemodialysis patients and 10 healthy controls were enrolled in a cross-sectional study. Using multiparameter flow cytometry, HBV-reactive T cells were analyzed and functionally dissected based on granzyme B, interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and IL-4 expression. Importantly, HBV-reactive CD4+ T cells were detected not only in all patients with sufficient titers but also in 70% of non-responders. Furthermore, a correlation between the magnitude of HBV-reactive CD4+ T cells and post-vaccination titers was observed. In summary, our data showed that HBV-reactive polyfunctional T cells were present in the majority of hemodialysis patients even if humoral immunity failed. Further studies are required to confirm their in vivo antiviral capacity. The ability to induce vaccine-reactive T cells paves new ways for improved vaccination and therapy protocols.

Autoimmunity-Related Risk Variants in PTPN22 and CTLA4 Are Associated With ME/CFS With Infectious Onset.

Single nucleotide polymorphisms (SNP) in various genes have been described to be associated with susceptibility to autoimmune disease. In this study, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients and controls were genotyped for five immune gene SNPs in tyrosine phosphatase non-receptor type 22 (PTPN22, rs2476601), cytotoxic T-lymphocyte-associated protein 4 (CTLA4, rs3087243), tumor necrosis factor (TNF, rs1800629 and rs1799724), and interferon regulatory factor 5 (IRF5, rs3807306), which are among the most important risk variants for autoimmune diseases. Analysis of 305 ME/CFS patients and 201 healthy controls showed significant associations of the PTPN22 rs2476601 and CTLA4 rs3087243 autoimmunity-risk alleles with ME/CFS. The associations were only found in ME/CFS patients, who reported an acute onset of disease with an infection (PTPN22 rs2476601: OR 1.63, CI 1.04-2.55, p = 0.016; CTLA4 rs3087243: OR 1.53, CI 1.17-2.03, p = 0.001), but not in ME/CFS patients without infection-triggered onset (PTPN22 rs2476601: OR 1.09, CI 0.56-2.14, p = 0.398; CTLA4 rs3087243: OR 0.89, CI 0.61-1.30, p = 0.268). This finding provides evidence that autoimmunity might play a role in ME/CFS with an infection-triggered onset. Both genes play a key role in regulating B and T cell activation.

MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes.

Proinflammatory type 1 T helper (Th1) cells are enriched in inflamed tissues and contribute to the maintenance of chronic inflammation in rheumatic diseases. Here we show that the microRNA- (miR-) 31 is upregulated in murine Th1 cells with a history of repeated reactivation and in memory Th cells isolated from the synovial fluid of patients with rheumatic joint disease. Knock-down of miR-31 resulted in the upregulation of genes associated with cytoskeletal rearrangement and motility and induced the expression of target genes involved in T cell activation, chemokine receptor- and integrin-signaling. Accordingly, inhibition of miR-31 resulted in increased migratory activity of repeatedly activated Th1 cells. The transcription factors T-bet and FOXO1 act as positive and negative regulators of T cell receptor (TCR)-mediated miR-31 expression, respectively. Taken together, our data show that a gene regulatory network involving miR-31, T-bet, and FOXO1 controls the migratory behavior of proinflammatory Th1 cells.

Heterozygous Loss-of-Function Mutations in DLL4 Cause Adams-Oliver Syndrome.

Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the presence of aplasia cutis congenita (ACC) of the scalp vertex and terminal limb-reduction defects. Cardiovascular anomalies are also frequently observed. Mutations in five genes have been identified as a cause for AOS prior to this report. Mutations in EOGT and DOCK6 cause autosomal-recessive AOS, whereas mutations in ARHGAP31, RBPJ, and NOTCH1 lead to autosomal-dominant AOS. Because RBPJ, NOTCH1, and EOGT are involved in NOTCH signaling, we hypothesized that mutations in other genes involved in this pathway might also be implicated in AOS pathogenesis. Using a candidate-gene-based approach, we prioritized DLL4, a critical NOTCH ligand, due to its essential role in vascular development in the context of cardiovascular features in AOS-affected individuals. Targeted resequencing of the DLL4 gene with a custom enrichment panel in 89 independent families resulted in the identification of seven mutations. A defect in DLL4 was also detected in two families via whole-exome or genome sequencing. In total, nine heterozygous mutations in DLL4 were identified, including two nonsense and seven missense variants, the latter encompassing four mutations that replace or create cysteine residues, which are most likely critical for maintaining structural integrity of the protein. Affected individuals with DLL4 mutations present with variable clinical expression with no emerging genotype-phenotype correlations. Our findings demonstrate that DLL4 mutations are an additional cause of autosomal-dominant AOS or isolated ACC and provide further evidence for a key role of NOTCH signaling in the etiology of this disorder.

Antigen receptor-mediated depletion of FOXP3 in induced regulatory T-lymphocytes via PTPN2 and FOXO1.

Regulatory T-cells induced via IL-2 and TGFß in vitro (iTreg) suppress immune cells and are potential therapeutics during autoimmunity. However, several reports described their re-differentiation into pathogenic cells in vivo and loss of their key functional transcription factor (TF) FOXP3 after T-cell antigen receptor (TCR)-signalling in vitro. Here, we show that TCR-activation antagonizes two necessary TFs for foxp3 gene transcription, which are themselves regulated by phosphorylation. Although the tyrosine phosphatase PTPN2 is induced to restrain IL-2-mediated phosphorylation of the TF STAT5, expression of the TF FOXO1 is downregulated and miR-182, a suppressor of FOXO1 expression, is upregulated. TGFß counteracts the FOXP3-depleting TCR-signal by reassuring FOXO1 expression and by re-licensing STAT5 phosphorylation. Overexpressed phosphorylation-independent active versions of FOXO1 and STAT5 or knockdown of PTPN2 restores FOXP3 expression despite TCR-signal and absence of TGFß. This study suggests novel targets for stabilisation and less dangerous application of iTreg during devastating inflammation.

miR-148a is upregulated by Twist1 and T-bet and promotes Th1-cell survival by regulating the proapoptotic gene Bim.

Repeatedly activated T helper 1 (Th1) cells present during chronic inflammation can efficiently adapt to the inflammatory milieu, for example, by expressing the transcription factor Twist1, which limits the immunopathology caused by Th1 cells. Here, we show that in repeatedly activated murine Th1 cells, Twist1 and T-bet induce expression of microRNA-148a (miR-148a). miR-148a regulates expression of the proapoptotic gene Bim, resulting in a decreased Bim/Bcl2 ratio. Inhibition of miR-148a by antagomirs in repeatedly activated Th1 cells increases the expression of Bim, leading to enhanced apoptosis. Knockdown of Bim expression by siRNA in miR-148a antagomir-treated cells restores viability of the Th1 cells, demonstrating that miR-148a controls survival by regulating Bim expression. Thus, Twist1 and T-bet not only control the differentiation and function of Th1 cells, but also their persistence in chronic inflammation.

Diffuse angiopathy in Adams-Oliver syndrome associated with truncating DOCK6 mutations.

Adams-Oliver syndrome (AOS) is a rare malformation syndrome characterized by the presence of two anomalies: aplasia cutis congenita of the scalp and transverse terminal limb defects. Many affected individuals also have additional malformations, including a variety of intracranial anomalies such as periventricular calcification in keeping with cerebrovascular microbleeds, impaired neuronal migration, epilepsy, and microcephaly. Cardiac malformations can be present, as can vascular dysfunction in the forms of cutis marmorata telangiectasia congenita, pulmonary vein stenoses, and abnormal hepatic microvasculature. Elucidated genetic causes include four genes in different pathways, leading to a model of AOS as a multi-pathway disorder. We identified an infant with mild aplasia cutis congenita and terminal transverse limb defects, developmental delay and a severe, diffuse angiopathy with incomplete microvascularization. Whole-genome sequencing documented two rare truncating variants in DOCK6, a gene associated with a type of autosomal recessive AOS that recurrently features periventricular calcification and impaired neurodevelopment. We highlight an unexpectedly high frequency of likely deleterious mutations in this gene in the general population, relative to the rarity of the disease, and discuss possible explanations for this discrepancy.

Mutations in NOTCH1 cause Adams-Oliver syndrome.

Notch signaling determines and reinforces cell fate in bilaterally symmetric multicellular eukaryotes. Despite the involvement of Notch in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ, DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 5' region and three coding variants (c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.743-1 G>T) in an affected father and daughter. These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent developmental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families. We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or bony component and are caused by alterations in the Notch signaling pathway.

Lymphocyte signaling: regulation of FoxO transcription factors by microRNAs.

The Forkhead box O (FoxO) family of transcription factors is important for the maintenance of immunological homeostasis and tolerance by controlling the development and function of B and T lymphocytes. Because dysregulation in FoxO activity can result in chronic inflammation and autoimmunity, the transcriptional activity of FoxO proteins is tightly controlled and generally dependent on complex posttranslational modifications that lead either to their nuclear entry and subsequent activation or, alternatively, to their nuclear export. The phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt) axis represents the major pathway phosphorylating and thereby inactivating FoxO proteins. However, recent results have revealed an additional posttranscriptional mechanism of FoxO inactivation by microRNAs. The discovery of this molecular pathway may provide a new therapeutic avenue for the modulation of FoxO activity in immune-mediated diseases using either microRNA targeting antagomirs or synthetic microRNA mimics, a topic that is addressed in this review.