Induced pluripotent stem cell macrophages present antigen to proinsulin-specific T cell receptors from donor-matched islet-infiltrating T cells in type 1 diabetes

Aims/hypothesis Type 1 diabetes is an autoimmune disorder characterised by loss of insulin-producing beta cells of the pancreas. Progress in understanding the cellular and molecular mechanisms underlying the human disease has been hampered by a dearth of appropriate human experimental models. We previously reported the characterisation of islet-infiltrating CD4+ T cells from a deceased organ donor who had type 1 diabetes. Methods Induced pluripotent stem cell (iPSC) lines derived from the above donor were differentiated into CD14+ macrophages and tested for their capacity to present antigen to T cell receptors (TCRs) derived from islet-infiltrating CD4+ T cells from the same donor. Results The iPSC macrophages displayed typical macrophage morphology, surface markers (CD14, CD86, CD16 and CD11b) and were phagocytic. In response to IFNγ treatment, iPSC macrophages upregulated expression of HLA class II, a characteristic that correlated with their capacity to present epitopes derived from proinsulin C-peptide to a T cell line expressing TCRs derived from islet-infiltrating CD4+ T cells of the original donor. T cell activation was specifically blocked by anti-HLA-DQ antibodies but not by antibodies directed against HLA-DR. Conclusions/interpretation This study provides a proof of principle for the use of iPSC-derived immune cells for modelling key cellular interactions in human type 1 diabetes. Electronic supplementary material The online version of this article (10.1007/s00125-019-04988-6) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

In order to generate iPSCs, donor derived PBMCs were cultured for 7 days in StemSpan TM SFEM II (STEMCELL Technologies, (https://www.stemcell.com) containing StemSpan TM erythroid expansion supplement (STEMCELL Technologies). Erythroid progenitors were reprogrammed using a Cytotune TM -iPS 2.0 Sendai Reprograming kit (ThermoFisher Scientific). Transduced cells were plated on culture dishes seeded with irradiated mouse embryonic fibroblasts (MEFs), and maintained in human pluripotent stem cell medium containing Knockout DMEM/20%Knockout serum replacer supplemented with 50ng/mL of FGF2 [2]. Two resulting iPSC lines (AF1 and AF2) were expanded and assessed for the expression of stem cell markers using flow cytometry and subjected to karyotype analysis using the Infinium CoreExome-24 SNP arrays. Cell lines were adapted to feeder free conditions prior to initiating differentiation and were maintained in Essential 8™ Medium (ThermoFisher Scientific).
Immunofluorescence staining: Undifferentiated iPSCs were grown on glass coverslips. The coverslips were washed with 1 x PBS then fixed in 4% PFA for 10 minutes at room temperature. Following this, the coverslips were placed in blocking buffer (10% FBS + 0.1% Triton-X in 1x PBS) for 1 hour at room temperature. Primary antibodies (see ESM table 1 for catalogue numbers and dilutions) were diluted in blocking buffer and incubated overnight at 4ºC. Secondary antibodies (ESM table 1) were diluted in blocking buffer and incubated at room temperature for 1 hour. Fluoromount-G (ThermoFisher Scientific) was applied to the coverslips which were then mounted on slides. Slides were imaged using a Zeiss confocal LSM 780 inverted microscope (https://www.zeiss.com). Image analysis was performed using ImageJ software, Version 1.0, 1-6-2019 to 30-7-2019, https://imagej.net).

Generation of iPSC Macrophages:
Differentiation towards the monocyte macrophage lineage was essentially performed as described [3], with the variation that embryoid bodies were formed using rotational cultures [4]. Embryoid bodies were formed in media supplemented with 20 ng/ml recombinant human (rh) bone morphogenetic protein 4 (BMP4, R&D Systems, R&D systems, https://www.rndsystems.com), 40 ng/ml rh vascular endothelial growth factor (VEGF, PeproTech, https://www.peprotech.com) 40 ng/ml rh stem cell factor (SCF, PeproTech), 10 ng/ml rh Fibroblast growth factor 2 (FGF2 5ng/ml used for the first day) (PeproTech), 5 ng/nl rh activin A (R&D Systems) day and CHIR99021 0.2 µM (Tocris Biosciences, https://www.tocris.com)(set up medium). Media was changed on day 1 with set up medium lacking Activin A, and again on day 2, with set up medium lacking both Activin A and CHIR. After day 8, EBs were transferred onto matrigel-coated, 6-well plates at ~ 20 EBs/well in media supplemented with 20 ng/ml rh VEGF and 20 ng/ml SCF, 10 ng/ml FGF2, 25 ng/ml of rh interleukin-3 (IL-3) (Peprotech) and 50 ng/ml of macrophage colony stimulating factor (M-CSF) (Peprotech). 50 ng/ml rh FLT3 receptor ligand (FLT3L, PeproTech) and 25 ng/ml granulocyte monocyte colony stimulating factor (GM-CSF) was added to the above media from day 11 of culture. The appearance of monocytic cells was assessed by flow cytometric analysis for CD14 expression at differentiation day 14. Monocytic cells shed from the EBs were further matured in media supplemented with 25 ng/ml of IL-3, 50 ng/ml of M-CSF 50 ng/ml FLT3L and 25 ng/ml GM-CSF. Post CD14 sorting, CD14 + high cells of monocytic lineage were matured for 5-7 days in media containing 100 ng/ml of M-CSF (or 50 ng/ml of GM-CSF when specified) to yield macrophages, which were subsequently activated with 20ng/ml of IFNg prior to use in T cell assays.
Flow cytometry and cell sorting: Adherent macrophage cultures were detached using TrypLE select (Thermofisher) to yield single cell suspension. Cells were washed with PBS and then resuspended in 100 µl of FACS wash (PBS with 2% Fetal calf serum). Cells were labelled with fluorophoreconjugated antibodies at the dilutions specified in Supplementary Table 2 and the mixture incubated for 20 minutes on ice. The cell suspension was washed twice with FACS wash to remove unbound antibodies and resuspended in FACS wash containing 1 μg/ml propidium iodide. Flow cytometric analysis was performed using a BD Fortessa flow cytometer. Flow cytometry gates were set relative to samples labelled with an isotype control antibody. Cell sorting was performed using a FACS Aria cell sorter (BD BioSciences, https://www.bdbiosciences.com). See ESM Table 1 for antibodies used for flow cytometric analysis.

Characterisation of iPSC macrophages:
For flow cytometric analysis, adherent macrophage cultures were first detached using TrypLE select (ThermoFisher) to yield a single cell suspension. Labelling of cells for flow cytometry was performed as detailed above using the fluorophoreconjugated antibodies specified in Supplementary Table 1.
Characterisation of iPSC macrophages using Giemsa-May Grunwald staining: Cells were collected in PBS and centrifuged at 900rpm for 5 minutes using a Thermo Shandon Cytospin 4 (https://www.thermofisher.com). Slides were air dried for 30 minutes, stained with 50% May-Grünwald solution for 5 min, washed twice with distilled water, then stained with 5% Giemsa for 20 minutes and finally rinsed with distilled water. Slides were air dried overnight and mounted with cover slips using DPX. Images were taken using Zeiss Axiocam mounted on an Axiovert 200 inverted microscope. Characterisation of iPSC macrophages with phagocytosis assays: For main figure 1f, iPSC Macrophages were plated at 100,000 cells/well of a 12 well plate and incubated overnight at 37ºC to allow cells to adhere. pHrodo TM Red E. coli BioParticles ® Conjugate (ThermoFisher, cat # P35361), reconstituted to a concentration of 1mg/ml, were added to macrophage cultures and incubated for 1-2 hours at 37˚C. Cells were then harvested using TrypLE select (ThermoFisher) and analysed using a BD Fortessa flow cytometer (https://www.bdbiosciences.com). A separate sample of cells with the added bioparticles was kept on ice as a negative control. For ESM figure 1g, cells from late stage (week 3) GM-CSF and M-CSF supplemented macrophage cultures were harvested using TrypLE select and collected by centrifugation. Cells were resuspended in medium containing either CFSE-(See below) or pHRodo-labelled (ThermoFisher, cat # P35361) E.coli bioparticles in the presence or absence of 20 µM Cytochalasin D (Sigma, cat# C2618, (https://www.sigmaaldrich.com). Cells were incubated at 37ºC for 1 hour with mixing. Additional controls included cells incubated in the presence of bioparticles at 4ºC. Unbound particles were removed by washing cells 4 times with 4 mls of FACS wash (PBS with 2% Fetal Calf Serum). After the final wash, cell pellets were resuspended in FACS wash containing a PE-Cy7 conjugated anti-CD16 antibody (ESM table 1) and processed for flow cytometry as described below.
CFSE labelling of E.coli. CFSE labelled E.coli were generated by incubating a 10µl pellet of heat killed (65ºC, 15 minutes) DH10β E.coli in 1 µM CFSE (resuspended in PBS) at 37º for 10 minutes. Unbound CFSE was removed by washing 5 times with 1 ml of PBS. Labelled E.coli was resuspended in 1 ml of PBS prior to use T-cell line culture. The SKW3 T-cell line used in this study was maintained in RPMI 1640 supplemented with 10% FCS. Antigen presentation assays: T-cell activation assays were performed essentially as described previously [5]. Briefly, SKW3 A1.9 TCR T cells / CD4 + T cells were incubated with 20-30,000 iPSC Macrophages at a 1:1 ratio and the specified concentrations of a synthetic proinsulin peptide, cpeptide or islet extract for 24 hours. T cell activation as a response to antigen presentation was quantified by flow cytometric analysis of CD69 expression, an antigen specific early T cell activation marker [6]. Co-culture of T cell lines with an HLA-matched EBV transformed B lymphoblastoid line (EBV BLL) (KJ; HLA-DRB1*03:01, 04:04; DQB1*02:01, 03:02) was used as a positive control. HLA blocking experiments were performed as described below.
Synthetic proinsulin peptides, c peptide and islet extract: Synthetic Peptide 11 was synthesized by Purar Chemicals (Shanghai, China) and reconstituted in 40% acetonitrile, 0.5% acetic acid, and water to 5 mmol/L, aliquoted, and stored at -80°C. Full length C peptide (PI 33-63 ) was purchased from Abcam. Islet lysates were prepared as previously described [7]. The sequence of C-peptide is EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ, and the sequence of Peptide-11 is highlighted in yellow. The minimum epitope recognized by the TCR from clone A1.9 is shown in red text.
HLA specificity analysis: HLA restriction was determined in two ways. First, we tested the effect of blocking HLA engagement using monoclonal antibodies specific for HLA-DR (clone L243), and -DQ (clone SPV-L3), which were added to cultures at a final concentration of 5-10 µg/mL. We also generated macrophages from an iPSC line (PB001) from healthy donor which was not HLA matched to the T1DM donor [8] derived TCRs. These HLA mismatched iPSC macrophages were included as a negative control for subsequent T cell activation assays.   Macrophages as compared to the responses elicited with the use of the HLA matched EBV transformed B-lymphoblastoid line. All iPSC derived macrophages used in this panel were activated with 20ng/ml IFNg for 48 hours prior to use. d. The islet derived CD4+ T-cell clone can self-present the TCR specific peptide (peptide 11) and addition of GM-CSF matured IFNg treated iPSC Macrophages does not increase the level of T-cell activation over and above that induced by selfpresentation. The key identifying each condition is indicated to the right of panel d. e. CFSE dilution (proliferation) experiment showing that self-presentation is a relatively weak driver of proliferation and that activated macrophages alone are also capable of eliciting a strong proliferation response (but does not induce CD69 -see, d). The combination of peptide and activated macrophages provides the strongest proliferative stimulus.