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Nuclear factor 'κ-light-chain-enhancer' of activated B cells (NF-κB) signaling is a signaling pathway used by most immune cells to promote immunostimulatory functions. Recent studies have indicated that regulatory T cells (Treg) differentially integrate TCR-derived signals, thereby maintaining their suppressive features. However, the role of NF-κB signaling in the activation of human peripheral blood (PB) Treg has not been fully elucidated so far. We show that the activity of the master transcription factor forkhead box protein 3 (FOXP3) attenuates p65 phosphorylation and nuclear translocation of the NF-κB proteins p50, p65, and c-Rel following activation in human Treg. Using pharmacological and genetic inhibition of canonical NF-κB signaling in FOXP3-transgenic T cells and PB Treg from healthy donors as well as Treg from a patient with a primary NFKB1 haploinsufficiency, we validate that Treg activation and suppressive capacity is independent of NF-κB signaling. Additionally, repression of residual NF-κB signaling in Treg further enhances interleukin-10 (IL-10) production. Blockade of NF-κB signaling can be exploited for the generation of in vitro induced Treg (iTreg) with enhanced suppressive capacity and functional stability. In this respect, dual blockade of mammalian target of rapamycin (mTOR) and NF-κB signaling was accompanied by enhanced expression of the transcription factors FOXP1 and FOXP3 and demethylation of the Treg-specific demethylated region compared to iTreg generated under mTOR blockade alone. Thus, we provide first insights into the role of NF-κB signaling in human Treg. These findings could lead to strategies for the selective manipulation of Treg and the generation of improved iTreg for cellular therapy., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Purpose: NF-κB signaling is critically important for regulation of both innate and adaptive immune responses. While activation of NF-κB has been implicated in malignancies such as leukemia and lymphoma, loss-of-function mutations affecting different NF-κB pathway components have been shown to cause primary immunodeficiency disorders. Recently, haploinsufficiency of NF-κB1 has been described in three families with common variable immunodeficiency (CVID)., Methods and Results: We studied a patient with recurrent respiratory infections and bacterial parapharyngeal abscess. Immunological investigations revealed normal total B- cell numbers, but hypogammaglobulinemia, decreased frequencies of class-switched B cells and impaired T-cell proliferation. Targeted next-generation sequencing using a custom-designed panel comprising all known PID genes (IUIS 2014 classification) and novel candidate genes identified a novel heterozygous frameshift mutation in the NFKB1 gene leading to a premature stop codon (c.491delG; p.G165A*31). We could show that the mutation leads to reduced phosphorylation of p105 upon stimulation, resulting in decreased protein levels of p50. The further disease course was mainly characterized by two episodes of severe EBV-associated lymphoproliferative disease responsive to rituximab treatment. Due to disease severity, the patient is considered for allogeneic hematopoietic stem cell transplantation. Interestingly, the father carries the same heterozygous NFKB1 mutation and also shows decreased frequencies of memory B cells but has a much milder clinical phenotype, in line with a considerable phenotypic disease heterogeneity., Conclusions: Deficiency of NF-κB1 leads to immunodeficiency with a wider phenotypic spectrum of disease manifestation than previously appreciated, including EBV lymphoproliferative diseases as a hitherto unrecognized feature of the disease.