Aire and Foxp 3 Expression in a Particular Microenvironment for T Cell Differentiation

Objective: The thymus is the primary lymphoid organ responsible for T cell development and the establishment of central self-tolerance. Among thymic epithelial cells, thymic nurse cells (TNC) interact closely with immature thymocytes and constitute a special microenvironment for T cell differentiation and selection. In addition, TNC express neuroendocrine self-antigens such as oxytocin and insulin-like growth factor-2, whose intrathymic transcription is regulated by the autoimmune regulator gene/protein (Aire). Both effector and natural regulatory T cell (nTreg) lineages develop in the thymus, but the mechanisms leading to nTreg selection in the thymus are still unclear. Foxp3 is the most specific nTreg marker that is required for nTreg functional activity, but not for engagement into the Treg lineage. Aire has been suggested to be a potential factor implicated in this role. The objective of this study was to characterize Aire and Foxp3 expression in TNC/thymocyte complexes. Methods: Aire and Foxp3 expression was investigated by RT-qPCR in TNC/thymocyte complexes isolated by enzymatic digestion and sedimentation. Aire and Foxp3 proteins were located by confocal microscopy and specific immunocytochemistry. Results: Both Aire and Foxp3 transcripts were Received: April 15, 2008 Accepted after revision: July 11, 2008 Published online: December 15, 2008 Vincent Geenen University of Liege, Center of Immunology Institute of Pathology CHU-B23 BE–4000 Liege-Sart Tilman (Belgium) Tel. +32 4366 2350 , Fax +32 4366 9859 , E-Mail vgeenen@ulg.ac. be © 2008 S. Karger AG, Basel 1021–7401/09/0161–0035$26.00/0 Accessible online at: www.karger. com/nim Hansenne /Louis /Martens /Dorban / Charlet-Renard /Peterson /Geenen Neuroimmunomodulation 2009;16:35–44 36 physial family, represented by oxytocin (OT) as the dominant peptide, as well as vasopressin [5–9] . The product of the autoimmune regulator gene (Aire) is a transcription factor involved in controlling the expression of many tissue-restricted antigens by medullary TEC [10] . Aire deficient mice develop autoantibodies and lymphocyte infiltrates in several organs, leading to a spectrum of autoimmune diseases [11, 12] . Several Aire mutations are also described in a rare recessive congenital disease, the autoimmune polyendocrinopathy candidiasis and ectodermal dystrophy syndrome [13] . Aire deletion in mice is associated with reduced expression of many (but not all) self-antigens in the thymus. Among the Aire-dependent genes, the OT, IGF-2 and insulin genes are strongly repressed following the deletion of the gene [11, 14] . A particular microenvironment of the thymus is formed by 2 cell types, thymic nurse cells (TNC), a TEC subtype, and immature thymocytes [for a review, see 15, 16] . These complexes were first described in 1980 by Wekerle et al. [17] and are observed both after isolation and in thymic sections [18, 19] . In situ, these TNC/thymocyte complexes have mainly been described in the outer cortex, and TNC-associated thymocytes are internalized in vacuoles of TNC [20, 21] . The number of thymocytes engulfed in TNC varies from 2 to 200 and determines the size of the complex. It has been reported that TNC can support T cell development and selection [16] . Neuroendocrine self-antigens such as OT and IGF-2 are expressed by TNC [7, 22] , but, at present, nothing is known about the regulation of self-antigen expression in TNC or about Aire expression in TNC/thymocyte complexes. As mentioned above, the thymus is also responsible for nTreg generation. The mechanisms leading to the differentiation and selection of nTreg in the thymus are still largely unclear. The transcription factor Foxp3 is required for the suppressive activity of nTreg, but is not necessary for engagement into the nTreg lineage [23, 24] . This strongly suggests the intervention of other factors, and Aire has recently been suggested to be involved in this process [25] . The objective of this study was to investigate whether Aire and Foxp3 are expressed in TNC/thymocyte complexes isolated from murine thymi. We show here that Aire is mainly expressed by TNC, while Foxp3 is located in the nucleus of thymocytes engulfed in TNC. These observations support the notion of a link between these transcription factors, and suggest that TNC may also be involved in nTreg development. Materials and Methods Animals Balb/c mice were provided by the Animal Department of Liege University. They were kept under conventional conditions with free access to food and water. Female mice were 5 weeks old at the time of the experiments, which were performed with the approval of the local ethics committee. Isolation of TNC/Thymocyte Complexes TNC were isolated from Balb/c thymus according to the methods previously described [17, 26] . The first step was thymic dissociation by enzymatic digestions, and the second step was TNC isolation by sedimentation. For each experiment, 20 thymi from Balb/c mice were dissected, placed in sterile cold Dulbecco’s phosphate-buffered saline (DPBS; Lonza) supplemented with 5% FCS (Invitrogen) and sliced into small pieces. Thymus fragments were shaken for 5 min in 50 ml of sterile cold DPBS supplemented with 5% FCS. The supernatant containing thymocytes was removed, and fragments were submitted to 5 successive enzymatic digestions. The first 2 digestions were performed with 25 ml of dispase (1.2 U/ml; Roche Diagnostics)/DNase (0.02 mg/ml; Roche Diagnostics) solution for 20 min under agitation. The next 3 digestions were performed with 25 ml of collagenase (40 U/ml; Sigma-Aldrich)/DNase (0.02 mg/ml) solution for 20 min under agitation. The ‘starting fraction’, composed of a pool of 5 digestion supernatants in RPMI-1640 (Lonza) supplemented with streptomycin (100 g/ml; Lonza), penicillin (100 U/ml; Lonza) and L -glutamine (2 m M ; Lonza), was used to isolate TNC by sedimentation. Four runs of sedimentation at 1 g were performed. The ‘starting fraction’ was put on 10 ml of FCS (Invitrogen) for 20 min (step 1). The supernatant was removed and the pellet was used for the following sedimentations (step 2: 10 ml of FCS for 15 min; step 3: 10 ml of FCS for 15 min; step 4: 5 ml of FCS for 15 min). An enriched fraction of TNC was obtained, and the number of cells was estimated by the trypan blue exclusion test (Lonza). Isolation of Thymocytes, CD4+ T Cells and TEC Thymocytes and splenocytes from Balb/c mice were isolated by mechanical disruption, erythrocyte lysis (Hybrid-Max , Sigma-Aldrich) and filtration through a 70m cell strainer (BD Biosciences). CD4+ cells from splenocytes were isolated using MACS technology [CD4 (L3T4) Microbeads and an LS column, Miltenyi Biotec] according to the manufacturer’s instructions. TEC were isolated from Balb/c thymus by successive enzymatic digestions. Thymi were cut into small pieces, suspended in 5 ml of DPBS per thymus and stirred for 30 min at 4 ° C. Supernatant was removed, and 3 enzymatic digestions were performed with Hanks’ balanced salt solution (4 ml/thymus, 2 ml/thymus and 1 ml/thymus, respectively; Lonza, Belgium) supplemented with collagenase D (1 mg/ ml; Sigma-Aldrich) and DNase I (10 g/ml; Roche Diagnostics) for 12, 12 and 5 min, respectively, at 37 ° C. The last supernatant was treated with trypsin (0.25%; Lonza)/0.02% EDTA for 10 min at 37 ° C. The reaction was stopped with Iscove’s modified Dulbecco’s medium (Lonza) containing 10% FCS (Invitrogen). The number of cells was estimated by the trypan blue exclusion test. Fresh TEC were cultured for 1 week in Dulbecco’s modified Eagle’s Medium (Lonza) supplemented with penicillin (100 U/ml), streptomycin (100 g/ml), L -glutamine (2 m M ), sodium pyruvate