ADAP plays a pivotal role in CD4+ T cell activation but is only marginally involved in CD8+ T cell activation, differentiation, and immunity to pathogens.

The adhesion and degranulation promoting adaptor protein (ADAP) is a multifunctional scaffold involved in many different signaling pathways that are important for the function of T cells, including the inside-out and outside-in signaling of integrins, the activation of NF-κB, and the subsequent production of proinflammatory cytokines (e.g., IFN-γ and IL-2). Strikingly, despite its well-established role in T cells, previous studies did not distinguish between CD4+ and CD8+ T cells, and thus, it is unknown whether ADAP fulfills equally important functions in both T cell subsets. We show here that despite comparable ADAP expression levels in CD4+ and CD8+ T cells, their function is differentially dependent on ADAP. Whereas in vitro TCR-stimulation experiments revealed that activation, proliferation, and adhesion are severely compromised in CD4+ T cells lacking ADAP, their CD8+ counterparts are hardly affected by ADAP deficiency. Accordingly, antigen-specific in vivo stimulation of adoptively transferred CD8+ T cells during Listeria monocytogenes (Lm) and influenza A virus (IAV) infection revealed only moderate effects of ADAP deficiency in terms of CD8+ T cell activation, proliferation, and differentiation, which, however, did not impair pathogen-specific immunity. Thus, we show for the first time that ADAP fulfills different functions in CD4+ and CD8+ T cells, with CD8+ T cells being less dependent on ADAP. Our data identify ADAP as a potential molecular target for T cell subset-specific therapeutic interventions.

Atypical IκB proteins in immune cell differentiation and function.

The NF-κB/Rel signalling pathway plays a crucial role in numerous biological processes, including innate and adaptive immunity. NF-κB is a family of transcription factors, whose activity is regulated by the inhibitors of NF-κB (IκB). The IκB proteins comprise two distinct groups, the classical (cytoplasmic) and the atypical (nuclear) IκB proteins. Although the cytoplasmic regulation of NF-κB is well characterised, its nuclear regulation mechanisms remain marginally elucidated. However, work from recent years indicated that nuclear IκBs contribute significantly to the modulation of NF-κB-mediated transcription in the immune system. Here, we discuss the role of the atypical IκB proteins Bcl-3, IκBζ, IκBNS, IκBη and IκBL for the regulation of gene expression and effector functions in immune cells.

IκBNS regulates murine Th17 differentiation during gut inflammation and infection.

IL-17-producing Th17 cells mediate immune responses against a variety of fungal and bacterial infections. Signaling via NF-κB has been linked to the development and maintenance of Th17 cells. We analyzed the role of the unusual inhibitor of NF-κB, IκBNS, in the proliferation and effector cytokine production of murine Th17 cells. Our study demonstrates that nuclear IκBNS is crucial for murine Th17 cell generation. IκBNS is highly expressed in Th17 cells; in the absence of IκBNS, the frequencies of IL-17A-producing cells are drastically reduced. This was measured in vitro under Th17-polarizing conditions and confirmed in two colitis models. Mechanistically, murine IκBNS (-/-) Th17 cells were less proliferative and expressed markedly reduced levels of IL-2, IL-10, MIP-1α, and GM-CSF. Citrobacter rodentium was used as a Th17-inducing infection model, in which IκBNS (-/-) mice displayed an increased bacterial burden and diminished tissue damage. These results demonstrate the important function of Th17 cells in pathogen clearance, as well as in inflammation-associated pathology. We identified IκBNS to be crucial for the generation and function of murine Th17 cells upon inflammation and infection. Our findings may have implications for the therapy of autoimmune conditions, such as inflammatory bowel disease, and for the treatment of gut-tropic infections.

Atypical IκB proteins - nuclear modulators of NF-κB signaling.

Nuclear factor κB (NF-κB) controls a multitude of physiological processes such as cell differentiation, cytokine expression, survival and proliferation. Since NF-κB governs embryogenesis, tissue homeostasis and the functions of innate and adaptive immune cells it represents one of the most important and versatile signaling networks known. Its activity is regulated via the inhibitors of NF-κB signaling, the IκB proteins. Classical IκBs, like the prototypical protein IκBα, sequester NF-κB transcription factors in the cytoplasm by masking of their nuclear localization signals (NLS). Thus, binding of NF-κB to the DNA is inhibited. The accessibility of the NLS is controlled via the degradation of IκBα. Phosphorylation of the conserved serine residues 32 and 36 leads to polyubiquitination and subsequent proteasomal degradation. This process marks the central event of canonical NF-κB activation. Once their NLS is accessible, NF-κB transcription factors translocate into the nucleus, bind to the DNA and regulate the transcription of their respective target genes. Several studies described a distinct group of atypical IκB proteins, referred to as the BCL-3 subfamily. Those atypical IκBs show entirely different sub-cellular localizations, activation kinetics and an unexpected functional diversity. First of all, their interaction with NF-κB transcription factors takes place in the nucleus in contrast to classical IκBs, whose binding to NF-κB predominantly occurs in the cytoplasm. Secondly, atypical IκBs are strongly induced after NF-κB activation, for example by LPS and IL-1ß stimulation or triggering of B cell and T cell antigen receptors, but are not degraded in the first place like their conventional relatives. Finally, the interaction of atypical IκBs with DNA-associated NF-κB transcription factors can further enhance or diminish their transcriptional activity. Thus, they do not exclusively act as inhibitors of NF-κB activity. The capacity to modulate NF-κB transcription either positively or negatively, represents their most important and unique mechanistic difference to classical IκBs. Several reports revealed the importance of atypical IκB proteins for immune homeostasis and the severe consequences following their loss of function. This review summarizes insights into the physiological processes regulated by this protein class and the relevance of atypical IκB functioning.

Cellular-FLIP, Raji isoform (c-FLIP R) modulates cell death induction upon T-cell activation and infection.

Dysregulation of apoptosis caused by an imbalance of pro- and anti-apoptotic protein expression can lead to cancer, neurodegenerative, and autoimmune diseases. Cellular-FLIP (c-FLIP) proteins inhibit apoptosis directly at the death-inducing signaling complex of death receptors, such as CD95, and have been linked to apoptosis regulation during immune responses. While the isoforms c-FLIPL and c-FLIPS are well characterized, the function of c-FLIPR remains poorly understood. Here, we demonstrate the induction of endogenous murine c-FLIPR in activated lymphocytes for the first time. To analyze c-FLIPR function in vivo, we generated transgenic mice expressing murine c-FLIPR specifically in hematopoietic cells. As expected, lymphocytes from c-FLIPR transgenic mice were protected against CD95-induced apoptosis in vitro. In the steady state, transgenic mice had normal cell numbers and unaltered frequencies of B cells and T-cell subsets in lymphoid organs. However, when challenged with Listeria monocytogenes, c-FLIPR transgenic mice showed less liver necrosis and better bacterial clearance compared with infected wild-type mice. We conclude that c-FLIPR expression in hematopoietic cells supports an efficient immune response against bacterial infections.

IκB(NS) protein mediates regulatory T cell development via induction of the Foxp3 transcription factor.

Forkhead box P3 positive (Foxp3(+)) regulatory T (Treg) cells suppress immune responses and regulate peripheral tolerance. Here we show that the atypical inhibitor of NFκB (IκB) IκB(NS) drives Foxp3 expression via association with the promoter and the conserved noncoding sequence 3 (CNS3) of the Foxp3 locus. Consequently, IκB(NS) deficiency leads to a substantial reduction of Foxp3(+) Treg cells in vivo and impaired Foxp3 induction upon transforming growth factor-ß (TGF-ß) treatment in vitro. Moreover, fewer Foxp3(+) Treg cells developed from IκB(NS)-deficient CD25(-)CD4(+) T cells adoptively transferred into immunodeficient recipients. Importantly, IκB(NS) was required for the transition of immature GITR(+)CD25(+)Foxp3(-) thymic Treg cell precursors into Foxp3(+) cells. In contrast to mice lacking c-Rel or Carma1, IκB(NS)-deficient mice do not show reduced Treg precursor cells. Our results demonstrate that IκB(NS) critically regulates Treg cell development in the thymus and during gut inflammation, indicating that strategies targeting IκB(NS) could modulate the Treg cell compartment.