The differentiation of Th17 cells is controlled by a complex network of transcription factors (TFs), including FOS and JUN proteins of the AP-1 family. The FOS-like proteins, FOSL1 and FOSL2 have recently been reported to control Th17 responses. The molecular mechanisms dictating their roles, however, are unclear. Moreover, although the functions of AP-1 TFs are largely governed by their protein-protein interactions, these are also poorly characterized in this milieu. Using affinity purification in combination with mass-spectrometry we established the first interactomes of FOSL1 and FOSL2 in human Th17 cells. In addition to their known interactions with JUN proteins, our analysis identified several novel binding partners of FOSL factors. Gene ontology analysis revealed RNA binding was enriched as the major functionality for FOSL1 and FOSL2 associated proteins, thereby suggesting possible mechanistic links that have not been studied before. Intriguingly, 29 interactors were found to be shared between FOSL1 and FOSL2, which included crucial regulators of Th17-fate. These findings, including unique and shared interactions, were validated using parallel reaction monitoring targeted mass-spectrometry (PRM-MS), with additional measurements with other laboratory methods. Overall, this study provides key insights into interaction-based signalling mechanisms of FOSL1 and FOSL2, which potentially control Th17 cell-development and associated pathologies

The digested peptides were spiked with synthetic peptide analogues of the validation targets and MSRT retention time peptides standards (Sigma). The samples were analysed by LC-MS/MS using an Easy-nLC 1200 coupled to Q Exactive HF mass spectrometer (Thermo Fisher Scientific). The peptides were loaded onto a 20 x 0.1 mm i.d. pre-column and separated with a 75 µm x 150 mm analytical column, both packed with 5 µm Reprosil C18 (Dr Maisch GmbH). A separation gradient from 8 to 39% B in 30 min was used at a flow rate of 300 nl/min (Solvent A: 0.1% formic acid in MiliQ H2O and Solvent B: 80% acetonitrile, 0.1% formic acid in MiliQ H2O). The data was acquired in a PRM mode with an isolation window setting of 1.6 m/z at a resolution of 15,000 for the Orbitrap, using a target AGC value of 50,000 and maximum injection time of 22ms.

Mononuclear cells were isolated from the human umbilical cord blood of healthy neonates (Turku University Central Hospital, Turku, Finland) using the Ficoll- Paque density gradient centrifugation (Ficoll-Paque PLUS; GE Healthcare). Naive CD4+ T cells were further purified using CD4+ Dynal positive selection beads (Dynal CD4 Positive Isolation Kit; Invitrogen), by following manufacturers protocol. CD4+ T-cells were activated with plate-bound α-CD3 (3.75 µg/ml; Immunotech) and soluble α-CD28 (1 μg/mL; Immunotech) in X-vivo 20 serum-free medium (Lonza). X-vivo 20 media was supplemented with L-glutamine (2 mM, Sigma-Aldrich) and antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin; Sigma-Aldrich). Th17-cell differentiation was induced using a cytokine cocktail of IL-6 (20 ng/mL; Roche), IL-1β (10 ng/mL) and TGF-β (10 ng/mL) in the presence of the neutralizing antibodies anti-IFN-γ (1 μg/mL) and anti-IL-4 (1 μg/mL) to block Th1 and Th2 polarization, respectively. For the control cells (Th0), CD4+ T-cells were TCR stimulated with α-CD3 and α-CD28, in the presence of neutralizing antibodies. All cytokines and neutralizing antibodies were purchased from R&D Systems, unless otherwise stated. All cultures were maintained at 37°C in a humidified atmosphere of 5% (v/v) CO2/air. Immunoprecipitation for FOSL1 and FOSL2 was performed using Pierce MS-Compatible Magnetic IP Kit (Thermo Fischer, Cat no.90409). 72h cultured Th17 cell pellets were lysed in appropriate volumes of cell-lysis buffer provided in the kit. FOSL1 (Santacruz Biotechnology, Cat no.sc-28310), FOSL2 (Cell Signaling Technology, Cat no.19967), mouse IgG (negative control for FOSL1; Cell Signaling, Cat no. 5415) and rabbit IgG (negative control for FOSL2; Cell Signaling Technology, Cat no. 2729) antibodies were used for immunoprecipitating the respective protein complexes. All antibodies were pre-incubated with protein A/G beads for 4–5h to form antibody-bead complexes. Lysates were first pre-cleared with control IgG-bead complexes, for 3h. Pre-cleared lysates were then incubated overnight with FOSL1/FOSL2 antibody-bead complexes (test IP) or corresponding control IgG-bead complexes (negative IP control). The pull-down fractions were washed (following manufacturer’s protocol) and eluted with appropriate volume of elution buffer. The eluted protein was either vacuum dried for MS analysis or run for western blotting. The IP eluates for IgG, FOSL1 and FOSL2 were denatured with urea buffer (8 M urea, 50mM Tris-HCl pH 8.0), followed by reduction using dithiothreitol (10 mM) at 370C for 1 h. The reduced cysteine residues were subsequently alkylated using iodoacetamide (14 mM, in darkness) at room temperature for 30 min. The samples were diluted to reduce the urea concentration (<1 molar), followed by digestion with sequencing grade modified trypsin at 37oC overnight (16-18 hours). The digested peptides were acidified and then desalted using C18 Stage Tips, prepared in-house using Empore C18 disks (3M, Cat No 2215). The desalted samples were dried in a SpeedVac (SAVANT SPD1010, Thermo Scientific) and then stored at -80oC until further analysis. For validation measurements, synthetic isotopic analogues (lysine 13C6 15N2 and arginine 13C6 15N4) were obtained for unique peptides from selected protein targets identified in the AP-MS discovery data (Thermo Fischer Scientific). The same sample preparation procedure was used for the validation experiments, with the exception that the samples were spiked with isotope-labelled peptides and MSRT retention time peptides standards (Sigma), prior to MS analysis.