Supplementary File 1. Methods.

Gene expression of isolated B cells

PBMCs were freshly isolated from blood by Ficoll-Paque density centrifugation. Subsequently, CD19+ B cells were isolated by MACS following the manufacturer’s instructions (Miltenyi Biotec) using positive selection using bead-labelled anti-CD19 antibodies, with a purity of 97±3%. Isolated cells were lysed in RLT+ (Qiagen) with β-mercaptoethanol and stored at -80°C until further use. RNA was extracted using the Allprep universal kit (Qiagen) according to the manufacturer’s instructions. Subsequently cDNA was created using the Biorad iScript kit. Quantitative Polymerase Chain Reaction (qPCR) was performed on a Quantstudio QPCR apparatus, with Taqman Beadchip technology (Applied Biosystems) under conditions as specified by the manufacturer. As housekeeping genes GUSB and GAPDH were included. Expression of the following mTOR pathway-related genes was assessed: MTOR (Hs00234508_m1), RPTOR (Hs00375332_m1), RICTOR (Hs00380903_m1), DEPTOR (Hs00961900_m1), AKT1 (Hs00178289_m1), IGF1R (Hs00609566_m1), IGF1 (Hs01547656_m1) and PTEN (Hs02621230_s1). Technical validation of RPTOR (Hs00375332_m1) and IGF1R (Hs00609566_m1) expression was performed by Taqman single gene qPCR assays. Expression was normalized to GUSB (Hs00939627_m1) and GAPDH (Hs02758991_g1) and subsequently was calculated as a fold change relative to the mean of the healthy control group set at 1, using the delta-delta CT method.

Immunofluorescence

To study local mTOR activation in salivary glands, immunofluorescence staining was performed on frozen salivary gland tissue sections from pSS and nSS patients using mouse monoclonal anti-CD20 (L26, Dako), anti-CD138 (MI15, Dako) anti-CD3 (F7.2.38, Dako) and anti-CCR9 (MAB1791, R&D) and rabbit anti-pS6 monoclonal antibody (Ser235/236, D57.2.2E, XP #4858, CellSignaling). Fluorescent labelled secondary antibodies were used: goat anti-mouse AF488 and goat anti-rabbit AF555 (Life Technologies). To assess specificity of the anti-pS6 antibody, as a negative control we followed the same protocol leaving out the primary antibody for pS6. No staining other than CD3, CD138, CD20 or CCR9 was observed (not shown). The surface area of each section was determined to quantify the number of positive cells per mm2, using a magnification of 400x, the mean size of the biopsies was 8mm2.

Flow cytometry

To analyse activation of mTORC1 in different cell subsets, antibodies against CD45 (PerCP, HI30, BioLegend), CD19 (BV605, SJ25C1, BD), CD20 (PE-Cy7, B9E9 Beckman Coulter), CD27 (PE, O323, Sony Biotechnology, APC-ef780 O323, eBioscience) , CD38 (APC, HIT2, eBioscience), IgG (FITC, Southern Biotec), CD3 (AF700, UCHT1, Sony Biotechnology), CD4 (BV711, OKT4, Sony Biotechnology), CD14 (BV785, M5E2, BioLegend) and CCR9 (PE, 248621, Bio-Techne) were used. Fresh PBMCs were used, which were first stained with Fixable Viability Dye (eBioscience), after which antibodies for extracellular staining were used, followed by fixation and permeabilization and staining with intracellular antibodies. The eBioscience Fixation/Permeabilization kit (#00-5123-43 and #00-5223-56) was used for intracellular staining and isotype control staining was performed (IgG1-FITC, REA control, Miltenyi Biotec). Activation of mTORC1 was assessed by intracellular detection of phosphorylated S6 ribosomal protein (pS6) expression (anti-S6 pS240 FITC-conjugated, clone REA420, Miltenyi Biotec), indicating kinase (S6K) activity downstream of mTORC1. Viability of PBMCs after culture experiments was assessed by Fixable Viability Dye (eBioscience) staining and proliferation of T and B cells was assessed using CellTrace Violet (ThermoFisher, Life Technologies), both according to manufacturer’s instructions.

Cell culture

Peripheral blood mononuclear cells (PBMCs, 0.5x10⁶ per well in 48 well plates) from healthy controls (n=2-7) and pSS patients (n=2-8) were cultured in the presence of different concentrations of rapamycin (1-100 nM, Sellek Chemicals) to study the effect of mTORC1 inhibition on T and B cell proliferation (6 days) and production of IFN-γ and IgG (9 days). mTOR pathway activation in B cells was done by BCR crosslinking using anti-IgM F(ab’)₂ fragments (Jackson ImmunoResearch Laboratories). To mimic the activation of lymphocytes observed in pSS patients, T and B cell activation was induced with a combination of superantigen Staphylococcal Enterotoxin B (SEB, 0.1ng/mL) and a TLR9 agonist (CpG-C, 3.3μg/mL). IgG production was measured in culture supernatants by ELISA (Bethyl Laboratories) and IFN-γ concentrations were assessed by Luminex.