Background/Purpose: The transcription factor Autoimmune Regulator (AIRE) is crucial for the establishment of central tolerance in the thymus. Recently, peripheral CD45⁺ extrathymic AIRE-expressing cells (eTACs) have been identified, which share features with antigen presenting cells, such as dendritic cells (DCs). Although, nuclear factor (NF)-κB signaling has been implicated in thymic AIRE expression, stimuli and pathways that induce extrathymic AIRE expression are hitherto unknown. We aimed to unravel the molecular mechanisms underlying the regulation of extrathymic AIRE expression in eTACs and DCs.

Methods: Confocal imaging was performed on secondary lymphoid tissues, including tonsils and lymph nodes to identify and characterize eTACs. Second, we employed a novel sorting strategy to isolate DCs and eTACs from tonsil tissue for RNA-sequencing (seq) to generate transcription profiles. To study the regulation of AIRE, monocyte-derived DCs (moDCs), DCs and eTACs were stimulated by anti-CD40 to induce NF-κB activation. Gene transcription and protein expression levels of AIRE, indoleamine-2,3-dioxygenase (IDO), programmed death ligand-1 (PD-L1) and non-canonical NF-κB signalling components were detected by real time-PCR, flow cytometry, Western blot and/or confocal microscopy. Activation of NF-κB signalling was modulated by siRNA-mediated silencing or using a small molecule inhibitor (SMI) of activating kinases.

Results: In human tonsil and lymph node tissue AIRE⁺ cells were identified, which displayed DC characteristics, including MHCII and CD40, but were relatively low for CD11c. Bioinformatic analysis on RNA-seq derived gene expression profiles of sorted eTACs and DCs from tonsil tissue revealed an increased expression of AIRE and immunoregulatorygenes, including IDO and PD-L1 in eTACs compared to DCs. In addition, the expression of genes implicated in non-canonical NF-κB signaling, including NIK and CD40, were significantly higher in eTACs compared to DCs. CD40 stimulation has been shown to be critical for IDO production by moDCs. CD40-stimulation of moDCs resulted in the activation of non-canonical NF-κB signaling, which was accompanied by upregulation of AIRE, IDO and PD-L1 expression. Interestingly, the induction of AIRE expression was abrogated by siRNA-mediated silencing of NF-κB-inducing kinase (NIK) . Likewise, CD40 stimulation of sorted eTACs and DCs resulted elevated AIRE, IDO and PD-L1 expression levels. Subsequently, modulation of NIK via an SMI resulted in the abrogation of AIRE, IDO and PD-L1 expression in eTACs and DCs. In contrast, specific modulation of canonical NF-κB signaling had no significant effects on AIRE, IDO and PD-L1 expression in CD40-stimulated eTACs and DCs.

Conclusion: Collectively, our data demonstrate that AIRE expression, as well as the expression of the immunoregulatory molecules IDO and PD-L1 in eTACs and DCs can be induced through CD40 stimulation in a non-canonical NF-κB signaling dependent manner. These findings concerning the regulation of AIRE and immunoregulatory molecule expression may point towards a novel role of eTACs in peripheral tolerance.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/non-canonical-nf-%ce%bab-signalling-is-required-for-extrathymic-aire-expression-and-immunoregulatory-molecules-in-cells-of-the-dendritic-lineage/