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

Use of tissue donor material was approved by the St Vincent’s Hospital Human Research Ethics Committee (approval no. SVH HREC-A 011/04). iPSC generation, growth and differentiation were approved by the Royal Children’s Hospital human research ethics committee (approval no. 33001A).

iPSC were generated from cryopreserved peripheral blood mononuclear cells (PBMCs) from a type 1 diabetic donor, using a previously described method [7]. Two resulting iPSC lines (‘AF1’ and ‘AF2’) were expanded and characterised further (see ESM Methods for details).

Undifferentiated iPSCs were examined for expression of sex determining region Y-box 2 (SOX2), octamer-binding protein 4 (OCT4) and E-cadherin (ECAD) using immunofluorescence analysis, as detailed in the ESM Methods (see ESM Table 1 for antibody details).

iPSCs were differentiated towards the monocyte/macrophage lineage using a protocol based on that reported by Yanagimachi et al. [8]. Macrophage maturation was performed using macrophage colony stimulating factor (M-CSF) or granulocyte monocyte colony stimulating factor (GM-CSF), as specified, following which macrophages were activated using IFNy prior to their use in T cell assays (see ESM Methods for further details).

Macrophages were characterised by flow cytometry using antibodies against established surface markers (ESM Table 1) and using May–Grünwald–Giemsa-stained cytospin preparations. Phagocytic activity was determined using a flow cytometry-based fluorescent bioparticle uptake assay (pHrodo Red Escherichia coli BioParticles [ThermoFisher, catalogue no. P35361] or carboxyfluorescein succinimidyl ester (CFSE)-labelled E. coli; see ESM Methods), whilst functionality was established using antigen presentation assays, as previously described [9].

Isolation of islet-infiltrating T cells underpinning this study was conducted as previously described [6]. In order to facilitate analysis of T cell receptor (TCR) engagement, sequences encoding TCRs from the CD4⁺ T cell clone A1.9 were cloned into the pRRLSIN lentiviral expression vector (pRRLSIN.cPPT.PGK-GFP.WPRE [Addgene; catalogue no. 12252; http://​n2t.​net/​addgene:​12252; RRID: Addgene_12,252;www.​addgene.​org] was a gift from D. Trono) and transduced into a derivative of the T cell leukaemia line SKW3. The resultant line is herewith referred to as SKW3 A1.9 TCR T cells (see ESM Methods for further details). SKW3 A1.9 TCR T cells/CD4⁺ T cells were incubated with iPSC macrophages at a 1:1 ratio and with a synthetic proinsulin peptide (herewith referred to as ‘peptide 11’), C-peptide or islet extract for 24 h. The sequence of C-peptide is EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ, and the sequence of peptide 11 is LQVGQVELGGGPGAGSLQ. The minimum epitope recognised by the TCR from clone A1.9 is VELGGGPGA (see ESM Methods).

T cell activation as a response to antigen presentation was quantified by flow cytometric analysis of CD69 expression. Co-culture of T cell lines with an HLA-matched Epstein-Barr Virus transformed lymphoblastoid B cell line (EBV-BLL) was used as a positive control. Further details are provided in ESM Methods, as well as details of the antigens used.

HLA restriction was determined using blocking monoclonal antibodies against HLA-DR and HLA-DQ (clone L243 and clone SPV-L3, respectively; www.​wehi.​edu.​au/​about-structure/​laboratory-operations/​antibody-services), and by employing antigen presenting cells (APCs) with a known mismatch at the HLA class II loci as a negative control (from iPSC line PB001, from a healthy donor [HLA-DQ8⁻]) (see ESM for details).

CFSE proliferation assays were performed as previously described [10] and as detailed in the ESM Methods. Briefly, CD4⁺ T cells were labelled with CFSE and a subpopulation of uniformly CFSE-labelled cells were isolated by FACS. They were then cultured with iPSC macrophages with or without antigen for 4 days and then analysed for retention of CFSE labelling by flow cytometry.

The iPSCs generated in this study possessed PSC-like morphology (ESM Fig. 1a) and demonstrated robust expression of the stem cell surface markers epithelial cell adhesion molecule (EPCAM) and CD9 (Fig. 1a and ESM Fig. 1b; n = 1, analysed in duplicate) and transcription factors OCT4 and SOX2 (ESM Fig. 1c). A schematic summarising the protocol used for generating iPSC macrophages is shown in Fig. 1b. Flow cytometric analysis showed that iPSC macrophages expressed typical macrophage markers CD14, CD11b and CD86, with a substantial proportion showing expression of CD16 and HLA class II (Fig. 1c and ESM Fig. 1d; n = 1, analysed in duplicate). Under bright field microscopy, iPSC macrophages displayed a uniform morphology, whilst May–Grünwald–Giemsa-stained cytospin preparations revealed large mononuclear cells with the vacuolated cytoplasm typical of macrophages (Fig. 1d, e). iPSC macrophage function was assessed by testing the capacity of these cells to phagocytose fluorescent E. coli bioparticles. Flow cytometry analysis showed that iPSC macrophages were highly phagocytic, with >98% of cells showing uptake of E. coli bioparticles (Fig. 1f); this phagocytosis was reduced by the actin polymerisation inhibitor cytochalasin D (ESM Fig. 1f; data from two independent experiments using pHrodo-E. coli [n = 1] and CFSE-E. coli [n = 1]). Finally, treatment of cultures with IFNγ upregulated the expression of macrophage surface markers, particularly CD16 and HLA class II (AF1, Fig. 1g). This was also confirmed by independent experiments with AF2 (ESM Fig. 1e; n = 1) and by three independent experiments performed with AF1 and AF2 (ESM Fig. 1g, n = 3). Overall, these findings indicate that iPSC macrophages display typical features of similar cells isolated from in vivo sources, including surface marker expression and basic functional properties.

To assess the antigen-presenting capabilities of iPSC macrophages, we used a flow cytometric assay in which macrophages were co-cultured with T cell lines expressing a proinsulin-specific TCR derived from a previously characterised islet-infiltrating CD4⁺ T cell clone, A1.9 (SKW3 A1.9 TCR T cells) [6]. T cell activation was assessed by measuring antigen-dependent upregulation of CD69, an antigen-specific early T cell activation marker (Fig. 2a). Flow cytometry indicated that CD69 expression was upregulated in SKW3 A1.9 TCR T cells when co-cultured with iPSC macrophages in the presence of a TCR-specific peptide antigen (‘peptide 11’; Fig. 2b). In the case of iPSC macrophages matured with M-CSF, effective antigen presentation required prior treatment with IFNγ, whilst those matured with GM-CSF were able to present peptide without further activation (Fig. 2b,c), a difference that may relate to HLA class II expression (ESM Fig. 2a).

We also compared the antigen-presenting capacity of iPSC macrophages with HLA-DQ8-expressing EBV-BLL cells, as well as HLA class II mismatched iPSC macrophages generated from a stock iPSC line (Fig. 2c). In these experiments, CD69 induction was measured as fold change over CD69 expression of T cell–APC cultures in which antigenic peptide was absent. These experiments confirmed that IFNγ-treated iPSC macrophages derived from the syngeneic donor were effective at presenting antigen and were more potent than the EBV-BLL cells, whilst iPSC macrophages from an HLA-DQ8 negative donor failed to induce CD69 expression on SKW3 A1.9 TCR T cells in response to peptide 11 (also see ESM Fig. 2b, c).

We compared the induction of CD69 on target T cells in the absence or presence of HLA blocking antibodies directed against HLA-DR and HLA-DQ. We showed that HLA-DR blocking antibodies did not affect the level of CD69 induction on SKW3 A1.9 TCR T cells, whilst HLA-DQ blocking antibodies significantly reduced CD69 upregulation (Fig. 2d). These findings confirm our previous results indicating that activation of the A1.9 TCR is HLA-DQ dependent [6].

In order to examine whether iPSC macrophages had mature phagocytic and endocytic functionality, we tested their capacity to process heterogeneous protein mixtures, represented by islet cell extract [11], as well as full length C-peptide, and to activate SKW3 A1.9 TCR T cells. The findings indicated that iPSC macrophages could induce a similar level of CD69 upregulation in target T cells when provided with peptide 11, C-peptide or islet extract (Fig. 2e). This result was confirmed by examining the ability of iPSC macrophages to activate the primary CD4⁺ T cell clone A1.9 from which the A1.9 TCR was isolated. In this setting, iPSC macrophages, matured with either M-CSF or GM-CSF, processed and presented antigen to the A1.9 primary T cell clone, measured by CD69 upregulation (Fig. 2f). As expected, we also observed that the A1.9 CD4⁺ cell clone could efficiently present peptide 11, leading to its self-activation (ESM Fig. 2d). By contrast, self-activation was not observed in cultures provided with islet extract, presumably reflecting the inability of the T cells to take up and process complex mixtures (Fig. 2f). Self-activation did not lead to a profound proliferative response, as measured by CFSE dilution, whereas activation driven by iPSCs macrophages in the presence of antigen was predictably far more robust (ESM Fig. 2e). Tellingly, self-activation was not observed using the SKW3 A1.9 TCR T cell lines, which lack the appropriate HLA class II for antigen presentation, underlining the value of using genetically modified T cell lines for examining APC–T cell interactions.