Immunogenicity of human embryonic stem cell-derived beta cells
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To overcome the donor shortage in the treatment of advanced type 1 diabetes by islet transplantation, human embryonic stem cells (hESCs) show great potential as an unlimited alternative source of beta cells. hESCs may have immune privileged properties and it is important to determine whether these properties are preserved in hESC-derived cells.
We comprehensively investigated interactions of both innate and adaptive auto- and allo-immunity with hESC-derived pancreatic progenitor cells and hESC-derived endocrine cells, retrieved after in-vivo differentiation in capsules in the subcutis of mice.
We found that hESC-derived pancreatic endodermal cells expressed relatively low levels of HLA endorsing protection from specific immune responses. HLA was upregulated when exposed to IFNγ, making these endocrine progenitor cells vulnerable to cytotoxic T cells and alloreactive antibodies. In vivo-differentiated endocrine cells were protected from complement, but expressed more HLA and were targets for alloreactive antibody-dependent cellular cytotoxicity and alloreactive cytotoxic T cells. After HLA compatibility was provided by transduction with HLA-A2, preproinsulin-specific T cells killed insulin-producing cells.
hESC-derived pancreatic progenitors are hypoimmunogenic, while in vivo-differentiated endocrine cells represent mature targets for adaptive immune responses. Our data support the need for immune intervention in transplantation of hESC-derived pancreatic progenitors. Cell-impermeable macro-encapsulation may suffice.
KeywordsAllograft rejection Autoimmunity Beta cells Embryonic stem cells Transplantation
Antibody-dependent cellular cytotoxicity
Cytotoxic T lymphocyte
Elongation factor 1α
Human embryonic stem cell
hESC-derived endocrine cell
hESC-derived pancreatic endodermal cell
Membrane cofactor protein
Membrane attack complex-inhibitory protein
Peripheral blood mononuclear cell
Primary tubular epithelial cell
Beta cell replacement by islet transplantation can functionally cure long-standing type 1 diabetes but its implementation is limited by the lack of donor organs, loss of graft function over time and the side effects of obligatory immunosuppression. Alternative sources of beta cells could overcome the shortage of human islets, while novel protective strategies and/or the potential for immune privilege of an alternative source of beta cells may help to overcome the immunosuppressive burden.
Several alternative sources of beta cells are currently being explored, including human embryonic stem cells (hESCs) , proliferating beta cell lines , induced pluripotent stem cells [3, 4] and xenogeneic islets [5, 6]. hESC-derived beta cells are well on the way to clinical translation with recent publications on improved safety and scaling of a protocol being made by Kroon and colleagues [1, 7, 8].
After transplantation, hESC-derived beta cells face the challenges posed by the human immune system for the first time. Partial early loss of grafts through innate immune reactions may be overcome by transplanting more cells or by choosing an alternative site to the liver. However, both recurrent autoimmunity and alloreactive responses remain a persistent threat to transplanted human-derived beta cells despite immunosuppression [9, 10, 11]. Additionally, alloreactive responses provoked by a graft can be a risk for future transplantations [12, 13, 14]. Conversely, complete lack of immune interaction can leave cells vulnerable to infections or may invoke an innate immune attack by natural killer (NK) cells . We recently showed this for immortalised beta cell lines .
Embryonic stem cells have immune privileged properties and can resist alloreactive responses . However, differentiation of hESCs to other cell types can result in the loss of this immunological privilege [17, 18, 19, 20, 21]. Insights into the immunogenicity of the alternative sources of beta cells can help to guide the choice of immune-protective strategies and the relevant immune monitoring to be employed in clinical introduction. We therefore investigated the immunogenicity of hESC-derived beta cells and their progenitors to adaptive immune responses relevant in transplantation.
hESC-derived pancreatic endodermal cells (hESC-PEs) were prepared from the CyT49 cell line (HLA A1, A3, B44, B57) at ViaCyte (San Diego, CA, USA), cryopreserved and shipped to Brussels, as described . For each series of experiments, cells were thawed and cultured for 72 h in db-N50-K50-E50 medium . A fraction of the cells was sent to Leiden in db medium  and the remaining cells were transplanted in Brussels. Encapsulated grafts were prepared by loading 4 × 106 cells in a macro-device (ViaCyte) and were implanted in the subcutis of non-diabetic NOD/severe combined immunodeficiency (SCID) male mice (7–8 weeks old, NOD.CB17-Prkdcscid/J; Charles River, L’Arbresle, France). Implants were analysed in vivo and ex vivo as described . In vivo graft function was followed over 20 weeks through measuring plasma human C-peptide levels 15 min after intraperitoneal injection of glucose; at post-transplant week 20, all recipients were confirmed to have reached plasma human C-peptide levels above 1 ng/ml. Implants were resected at post-transplant week 20–25 and dispersed for analysis before sending to Leiden in phosphate-buffered Ham’s F-10 medium (Gibco, Bleiswijk, the Netherlands), supplemented with 0.5% human albumin, 2 mmol/l l-leucine and 2 mmol/l l-glutamine. Upon arrival, hESC-PEs and in vivo-differentiated cells were dissociated with 0.05% trypsin-EDTA 1X (Gibco) to single cells for immunological assays.
Human peripheral blood mononuclear cells (PBMCs) were separated from whole blood (for T cells) or buffy coats (for lymphocytes) by Ficoll–Hypaque density gradient. Peripheral blood lymphocytes were left over after CD14 depletion from PBMCs with CD14-MicroBeads according to the manufacturer’s protocol (Miltenyi Biotec, Auburn, CA, USA). Preproinsulin (PPI)-specific T cell clone 1E6 generation was described previously . Briefly, PBMCs from an individual with type 1 diabetes were stimulated with PPI15-24 peptide. PPI-specific cytotoxic T lymphocytes (CTLs) were sorted by FACS and expanded. Alloreactive CTL clones JS132 (to HLA-A2) and C776 (to HLA-A1) were generated by stimulating PBMCs, collected from a healthy donor, with irradiated Epstein–Barr virus (EBV)-transformed B cell line expressing the HLA of interest. After several rounds of stimulation and enrichment, the alloreactive population was cloned by limiting dilution at 0.5 cell/well . CTL clones to cytomegalovirus (CMV) peptides VTEHDTLLY in HLA-A1 (clone 3c8) and NLVPMVATV in HLA-A2 (clone 18) were generated by single cell sorting of CD8+ T cells stained with the respective tetramers and expanded using phytohaemagglutinin stimulation . HEK-293 and primary tubular epithelial cells (PTECs, line HK-2) were cultured in DMEM/F-12 medium (Invitrogen, Landsmeer, the Netherlands) supplemented with 2 mmol/l l-glutamine, 25 mmol/l HEPES, 50 U/ml penicillin and 50 μg/ml streptomycin (all purchased from Invitrogen) and, for PTECs, also 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium, 36 ng/ml hydrocortisone and 10 ng/ml epidermal growth factor (all purchased from Sigma, Zwijndrecht, the Netherlands). B lymphocytes (B-LCL) lines were cultured in IMDM (Invitrogen) supplemented with 2 mmol/l l-glutamine, 25 mmol/l HEPES, 50 U/ml penicillin, 50 μg/ml streptomycin and 5% FBS.
Human monoclonal antibodies recognising HLA-A68 or HLA-B8 were selected from a panel as described previously . In short, heterohybridomas were created by EBV transformation and cloning of B-LCL of multiparous women. The HLA specificities of the produced human monoclonal antibodies were validated using a complement-dependent cytotoxicity test on PBMCs.
Beta cell-specific T helper (Th) cell supernatant fractions were generated by incubating islet preparation reactive Th1 clone (1c6) from a diabetic patient with HLA-matched PBMCs pre-incubated with or without 10 μg/ml antigen in RPMI 1640 medium (Gibco) mixed without and with 11 mmol/l glucose to achieve 5.6 mmol/l d-glucose and supplemented with 2 mmol/l glutamine (Gibco) . After 3 days the supernatant fraction was harvested and frozen until use.
For compatibility with autoreactive CTL clone 1E6, beta cell line EndoC-βH1 was transduced with a lentiviral vector containing HLA-A02:01 under elongation factor 1α (EF1α) promotor at multiplicity of infection of 2. Third-generation self-inactivating lentivirus vectors were produced as described previously . The generation of human cell lines and antibodies was carried out after obtaining informed consent and with approval of the institutional review board, in accordance with the 2008 revised principles of the Declaration of Helsinki.
Alloreactive cellular cytotoxicity was assessed by chromium release (PerkinElmer, Waltham, MA, USA). Briefly, dispersed hESC-derived cells were labelled with 51Cr for 60 min, washed three times and incubated with alloreactive T cells or, for antibody-dependent cellular cytotoxicity (ADCC), human monoclonal antibodies and peripheral blood lymphocytes in different effector-to-target ratios for 4–6 h or overnight. 51Cr release in supernatant fractions was assessed on a WIZARD2 γ-counter (Perkin Elmer, Waltham, MA, USA). Specific lysis was calculated as [(experimental release − spontaneous release) / (max release − spontaneous release)] × 100%.
Complement-dependent cytotoxicity was assessed with hESC-derived cells as targets in a clinical cross match assay . In short, dispersed cells were incubated with serum containing alloreactive antibodies with known specificity and incubated in triplicate in Therasaki plates at room temperature for 1 h. Rabbit complement (Inno-train, Frankfurt am Main, Germany) was added and the plates were incubated for another hour. Cell lysis was assessed by adding propidium iodide–ink solution and measured on the Patimed system (Leica, Rijswijk, the Netherlands). Minimal and maximum lysis was set to HLA-antibody-negative serum cell death and parallel lysis of HLA-matched lymphocyte targets, respectively.
At 20 h post transduction, virus supernatant fraction was removed from HLA-A02:01 transduced cells by centrifugation. The cells were cultured in Ham’s F-10 medium (Gibco) supplemented with 200 mmol/l glutamine (Gibco), 0.5% BSA fraction V (Sigma-Aldrich, Zwijndrecht, the Netherlands), 1 mol/l CaCl2 and 1.8 g/l d-glucose for 4 days to allow transgene expression before trypsin dispersion and incubation with PPI-specific CTL clone 1E6 or CMV-specific clone 18 in a 1:5 ratio. After overnight incubation, cells were stained with Fixable Viability Dye eFluor 450 (eBioscience, Vienna, Austria), CD45-PerCP (2D1; BD, Breda, the Netherlands) and HLA-A2–fluorescein isothiocyanate (FITC) (BD), then fixed in paraformaldehyde and permeabilised with saponin before staining with guinea pig anti-insulin (Free University Brussels, Brussels, Belgium) and donkey anti-guinea pig Alexa 647 (Jackson Immunoresearch Laboratories, West Grove, PA, USA). Cells were measured on a CANTO II flow cytometer (BD). Transduction efficiency was determined by staining with anti-HLA-A2–Allophycocyanin (APC) (Bb7.2; BD) on a Calibur flow cytometer (BD).
Cell surface staining
Cell surface antigens were analysed by FACS on a BD FACSCalibur after 20 min staining at 4°C with antibodies to HLA class I–FITC (W6/32; BD), HLA-DR–FITC (L243; BD), IgG1–APC (MOPC-21; BD), IgG2a–FITC (G155-178; BD), IgG2a–Phycoerythrin (G155-178; BD), anti-CD46–APC (MEM-258; ImmunoTools, Friesoythe, Germany), anti-CD55–Phycoerythrin (IA10; BD) and anti-CD59–APC (OV9A2, eBioscience).
Data were excluded if positive or negative controls failed. Additional intermediate titrations were performed in parallel in most samples that were left out for clarity to the reader. These data supported the conclusion. Cell lines for comparison were available in our laboratory unless stated otherwise and mycoplasma contamination was regularly excluded. Non-commercial antibodies had been generated and validated in our department before  and were of IgG subclass.
Data are represented as mean and SE unless stated otherwise. GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA) was used to create graphs and perform analysis. Student’s t test was used to compare continuous data and Fisher’s exact test was used for binominal data; p < 0.05 was considered statistically significant. All immune assays were replicated three times.
To confirm peptide-specific killing of hESC-derived cells through their cognate HLA, we assessed killing by CTLs recognising CMV peptide in cognate HLA-A1. Similar to alloreactive CTLs, CMV-specific CTLs did not effectively kill hESC-PEs loaded with CMV peptide unless the hESC-PEs were exposed to IFNγ or the assay was prolonged. The differentiated hESC-ECs were vulnerable to alloreactive and CMV-specific CTLs without prior IFNγ treatment, while prolonged exposure increased killing to > 90% (Fig. 2).
Insights into the immunogenicity of beta cell-like cells from sources that are alternatives to human cadaver islets will help the decision-making process when choosing immune-protective strategies to test and the most relevant immune monitoring to perform in clinical translation. hESC-PEs are promising candidates for clinical application . We therefore investigated the immunological properties of hESC-derived pancreatic cell preparations before and after differentiation to endocrine cells. We found that hESC-PEs maintain immune privilege with low HLA expression, although upregulation appears possible in inflammatory conditions which then render the cells vulnerable to adaptive immune responses. In vivo differentiation of hESC-PEs into a preparation with pancreatic endocrine cells also leads to immunological differentiation with increasing HLA expression and sensitivity to adaptive immune responses and also increased resistance to complement-mediated attack. Recognition and destruction by autoreactive PPI-specific T cells verified mature beta cell immune presentation by hESC-ECs.
Low expression of HLA by hESCs and hESC-derived cells underlies the hypoimmunogenicity observed in most published studies, effectively hiding cells from T cell recognition and alloreactive antibodies, while differentiation can upregulate HLA and increase vulnerability to immunity [17, 18, 19, 20, 21]. In our study, immunogenicity to alloreactive and peptide (CMV)-specific responses was related to HLA expression. hESC-PEs expressed very low levels of HLA in a non-inflamed environment, making them hypoimmunogenic, while differentiation to hESC-ECs resulted in an increase in HLA expression and immunogenicity to T cells to levels reported for human islets in the literature [23, 31, 32, 33, 34].
Despite the hypoimmunogenicity of hESC-PEs, resistance was incomplete after prolonged exposure to high numbers of CTLs. This suggests that hESC-PEs do not employ active suppression of CTL responses, as has been observed in other studies . Further, HLA expression could be induced by mimicking inflammation with IFNγ. While this increased immunogenicity to alloreactive T cells and antibodies, the incomplete immune privilege may help to ensure adequate virus control in case of infection and prevent destruction by inflammation-activated NK cells .
Increased expression of HLA through inflammation or differentiation of hESC to beta cells may make cells vulnerable to recurrent autoimmunity if cells are transplanted to an HLA-matched individual with type 1 diabetes. After realising HLA match through transduction of in vivo-differentiated hESC-ECs, the insulin-expressing cells could be selectively eliminated by autoreactive CTLs previously isolated from a donor with type 1 diabetes . Thus, we show hESC-derived beta cells risk recurrent autoimmunity in an HLA-matched setting. Further, by proving presentation of the PPI peptide recognised by the CTL clone, we verify that hESC-ECs have active cellular mechanisms in place to ensure immune surveillance and that they display genuine insulin production allowing natural presentation of PPI epitopes in HLA.
In vivo differentiation of hESC-PEs to hESC-ECs increased resistance to complement-mediated attack despite increased expression of antigen (HLA) recognised by alloreactive antibodies. Protection from complement is mediated through complement inhibitory receptors, such as CD46 (MCP), CD55 (DAF) and CD59 (MIP), which are known to be expressed by human islets [36, 37]. All three receptors were expressed by the hESC-derived cells but CD55 expression was low on hESC-PEs, which may explain their vulnerability to complement-mediated cytotoxicity.
Although we were able to study diverse immune mechanisms relevant for transplantation of alternative beta cells, extrapolation of these results to the clinical transplantation setting is limited by several factors. First, animal models for in vivo differentiation of hESC-PEs to hESC-ECs may not completely reflect the differentiation process in humans. Second, culture conditions that are required for investigation of explanted in vivo-differentiated cells may alter the properties of the cells or the composition of the population. To this end, staining for beta cells did not show selective reduction of beta cells. Incomplete dissociation of naturally aggregating hESC-derived pancreatic endodermal cells and endocrine cells required for immune assays may contribute to the variation and relatively large standard error in the cytotoxicity assays (Figs 2, 4).
A head-to-head comparison with primary human islet cells would be of interest and was considered when setting up this study. However, it proved to be logistically impossible. hESCs require 4 months of in vivo differentiation into endocrine cells and the ex vivo experiments must be started within 24 h after explantation to assure optimal cell quality. The supply of fresh human islets for research purposes is highly infrequent and rare, precluding parallel testing with differentiated hESC-ECs. This was limited further by the HLA restrictions enforced by this type of experiment. We therefore looked to data on beta cell immunogenicity from the literature for the purpose of comparison [23, 31, 32, 33, 34, 36].
The identified immunogenicity of hESC-derived pancreatic endocrine cells highlights the need for an immune suppressive strategy for human transplantation. The vulnerability of these hESC-derived cells to cellular attack and their resistance to complement-mediated attack suggests that providing protection by cell-impermeable macro-encapsulation could be a successful strategy in clinical implementation. Alternatively, or additionally, the initial hypoimmunogenicity of the engrafted pancreatic endoderm may provide a window of opportunity in which to induce graft-specific tolerance.
In conclusion, pancreatic endoderm progenitors maintain hypoimmunogenicity, while differentiation of hESCs to pancreatic endocrine cells increases vulnerability to cellular immunity. Inflammatory conditions further increase immunogenicity. Yet, maturation of pancreatic endoderm to endocrine cells enforces resistance to complement-mediated cytotoxicity. This implies that, while simply preventing inflammation around transplantation of pancreatic endoderm may suffice initially, the maturing graft may need protection from cellular immune attack. Therefore, protecting grafts by cell-impermeable macro-encapsulation could be a successful strategy in clinical implementation. Alternatively, or in addition, the hypoimmunogenicity of the initially engrafted pancreatic endoderm may create a window of opportunity in which to induce graft-specific tolerance.
The authors thank the European Commission and the JDRF for supporting this work and thank R. J. Lebbink (Medical Microbiology, University Medical Center Utrecht, Utrecht, the Netherlands) for the HLA-A2 expression vector and A. Mulder (IHB, LUMC) for providing human HLA specific antibodies.
This work was funded by the European Commission (BetaCellTherapy, no. 241883 in the FP7 program) and the JDRF (17-2013-296).
Duality of interest
LM, EK and EPB are employees of ViaCyte, receiving salary and stock options. DP is a member of ViaCyte’s scientific and clinical advisory board. All other authors declare that there is no duality of interest associated with their contribution to this article.
The study was conceived and designed by CRT, DP and BOR, with MP, LM, EK and EPB also being responsible for the design. AZ, MP, GS, LM, EK, EPB and DP provided study material. CRT, AZ, GD, SHB-S and GS were responsible for collection and/or assembly of data and data analysis and interpretation. BOR was responsible for analysis and interpretation of data. CRT, AZ, LM, EK, EPB, DP and BOR wrote the manuscript and GD, SHB-S, MP and DP revised it. All authors approved the final version of the manuscript. BOR supervised the study and is the guarantor of this work.
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