Abnormal biomarkers predict complex FAS or FADD defects missed by exome sequencing.

BACKGROUND: Elevated TCRαß+CD4-CD8- double-negative T cells (DNT) and serum biomarkers help identify FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified on standard exon sequencing (ALPS-undetermined: ALPS-U). OBJECTIVE: We sought to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. METHODS: Genetic analysis included whole FAS gene sequencing, copy number variation analysis, and sequencing of FAS cDNA and other FAS pathway-related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss of heterozygosity (sLOH). RESULTS: Nine of 16 patients with ALPS-U lacked FAS expression on CD57+DNT predicting heterozygous "loss-of-expression" FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7 of 9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT; 1 patient showed a FAS exon duplication. Three patients had reduced FAS expression, and 2 of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the 4 ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. CONCLUSION: A combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing.

Human RIPK1 deficiency causes combined immunodeficiency and inflammatory bowel diseases.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a critical regulator of cell death and inflammation, but its relevance for human disease pathogenesis remains elusive. Studies of monogenic disorders might provide critical insights into disease mechanisms and therapeutic targeting of RIPK1 for common diseases. Here, we report on eight patients from six unrelated pedigrees with biallelic loss-of-function mutations in RIPK1 presenting with primary immunodeficiency and/or intestinal inflammation. Mutations in RIPK1 were associated with reduced NF-κB activity, defective differentiation of T and B cells, increased inflammasome activity, and impaired response to TNFR1-mediated cell death in intestinal epithelial cells. The characterization of RIPK1-deficient patients highlights the essential role of RIPK1 in controlling human immune and intestinal homeostasis, and might have critical implications for therapies targeting RIPK1.

Distinct molecular response patterns of activating STAT3 mutations associate with penetrance of lymphoproliferation and autoimmunity.

Germline STAT3 gain-of-function (GOF) mutations have been linked to poly-autoimmunity and lymphoproliferation with variable expressivity and incomplete penetrance. Here we studied the impact of 17 different STAT3 GOF mutations on the canonical STAT3 signaling pathway and correlated the molecular results with clinical manifestations. The mutations clustered in three groups. Group 1 mutants showed altered STAT3 phosphorylation kinetics and strong basal transcriptional activity. They were associated with the highest penetrance of lymphoproliferation and autoimmunity. Group 2 mutants showed a strongly inducible transcriptional reporter activity and were clinically less penetrant. Group 3 mutants were mostly located in the DNA binding domain and showed the strongest DNA binding affinity despite a poor transcriptional reporter response. Thus, the GOF effect of STAT3 mutations is determined by a heterogeneous response pattern at the molecular level. The correlation of response pattern and clinical penetrance indicates a significant contribution of mutation-determined effects on disease manifestations.

Activated PI3Kδ syndrome type 2: Two patients, a novel mutation, and review of the literature.

BACKGROUND: Autosomal dominant gain-of-function mutations in PIK3R1 encoding for the regulatory subunit (p85α, p55α, and p50α) of Class IA phosphoinositide 3-kinase (PI3K) result in the activated PI3Kδ syndrome (APDS) type 2 characterized by childhood-onset combined immunodeficiency, lymphoproliferation, and immune dysregulation. To improve clinical awareness and understanding of these rare diseases, we reviewed all hitherto published cases with APDS type 1 and type 2 for their clinical and immunologic symptoms and added novel clinical, immunologic, and genetic findings of two patients with APDS type 2. METHODS: Clinical, immunologic, and genetic evaluation of two new patients with APDS2 was performed followed by the systematic collection of all available previously published data of patients with APDS1 and APDS2. RESULTS: Patients with APDS type 1 (n = 49) and type 2 (n = 15) showed an indistinguishable immunologic phenotype. Overlapping clinical features shared by APDS type 1 and type 2 were observed, but our review also revealed previously unnoticed clinical differences such as remarkably high incidence of microcephaly, poor growth/short stature in patients with APDS2. Clinical management and outcome were variable and included prophylactic antibiotics, immunosuppression, immunoglobulin substitution, and hematopoietic stem cell transplantation. CONCLUSIONS: A disease-specific registry collecting prospective and long-term follow-up data of patients with APDS, as currently set up by the European Society for Immunodeficiencies, are needed to better understand the natural history and to optimize treatment concepts and thereby improving the outcome of this heterogenous patient group.

A distinct CD38+CD45RA+ population of CD4+, CD8+, and double-negative T cells is controlled by FAS.

The identification and characterization of rare immune cell populations in humans can be facilitated by their growth advantage in the context of specific genetic diseases. Here, we use autoimmune lymphoproliferative syndrome to identify a population of FAS-controlled TCRαß+ T cells. They include CD4+, CD8+, and double-negative T cells and can be defined by a CD38+CD45RA+T-BET- expression pattern. These unconventional T cells are present in healthy individuals, are generated before birth, are enriched in lymphoid tissue, and do not expand during acute viral infection. They are characterized by a unique molecular signature that is unambiguously different from other known T cell differentiation subsets and independent of CD4 or CD8 expression. Functionally, FAS-controlled T cells represent highly proliferative, noncytotoxic T cells with an IL-10 cytokine bias. Mechanistically, regulation of this physiological population is mediated by FAS and CTLA4 signaling, and its survival is enhanced by mTOR and STAT3 signals. Genetic alterations in these pathways result in expansion of FAS-controlled T cells, which can cause significant lymphoproliferative disease.

High-throughput detection of nuclear factor-kappaB activity using a sensitive oligo-based chemiluminescent enzyme-linked immunosorbent assay.

Contemporary research on cellular signaling has undergone a shift of focus from qualitative measurements of single signaling pathways to high-throughput quantitation of comprehensive signaling networks. Notably, nuclear factor-kappaB (NFkappaB) is a family of transcription factors involved in immune and inflammatory responses, developmental processes, cellular growth and apoptosis and is deregulated in a number of disease states. We have established a chemiluminescent oligonucleotide-based enzyme-linked immunosorbent assay (co-ELISA) that is simple and quantitative. In contrast to currently used assays, it allows quantitation of all NFkappaB components (i.e., RelA, p50, p52, RelB and c-Rel). In addition, it can make use of whole extract and does not require cumbersome nuclear/cytosolic fractionation, saving time and resources. Co-ELISA has a 3.5- to 43-fold higher signal-over-noise ratio than currently available assays, whereas the percent relative standard deviation is 3- to 6-fold lower. Furthermore, the novel method is faster than electrophoretic mobility shift assay, not restricted to transfectable cells as is the case for luciferase reporter assays and 10 times more cost efficient than commercially available ELISA assays. Co-ELISA is a sensitive, fast and cost-efficient quantitation method for all DNA-binding NFkappaB proteins that can be used in high-throughput experimentation.

Long-term robustness of a T-cell system emerging from somatic rescue of a genetic block in T-cell development.

BACKGOUND: The potential of a single progenitor cell to establish and maintain long-term protective T-cell immunity in humans is unknown. For genetic disorders disabling T-cell immunity, somatic reversion was shown to support limited T-cell development attenuating the clinical phenotype. However, the cases reported so far deteriorated over time leaving unanswered the important question of long-term activity of revertant precursors and the robustness of the resulting T-cell system. METHODS: We applied TCRß-CDR3 sequencing and mass cytometry on serial samples of a now 18 year-old SCIDX1 patient with somatic reversion to analyse the longitudinal diversification and stability of a T-cell system emerging from somatic gene rescue. FINDINGS: We detected close to 105 individual CDR3ß sequences in the patient. Blood samples of equal size contained about 10-fold fewer unique CDR3ß sequences compared to healthy donors, indicating a surprisingly broad repertoire. Despite dramatic expansions and contractions of individual clonotypes representing up to 30% of the repertoire, stable diversity indices revealed that these transient clonal distortions did not cause long-term repertoire imbalance. Phenotypically, the T-cell system did not show evidence for progressive exhaustion. Combined with immunoglobulin substitution, the limited T-cell system in this patient supported an unremarkable clinical course over 18 years. INTERPRETATION: Genetic correction in the appropriate cell type, in our patient most likely in a T-cell biased self-renewing hematopoietic progenitor, can yield a diverse T-cell system that provides long-term repertoire stability, does not show evidence for progressive exhaustion and is capable of providing protective and regulated T-cell immunity for at least two decades. FUNDING: DFG EH 145/9-1, DFG SCHW 432/4-1 and the German Research Foundation under Germany's Excellence Strategy-EXC-2189-Project ID: 390939984.