Transplantation of autoimmune regulator-encoding bone marrow cells delays the onset of experimental autoimmune encephalomyelitis

Authors

  • Hyun-Ja Ko,

    1. Department of Immunology, Central Clinical School, Monash University, Melbourne, VIC, Australia
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    • The laboratories of these authors contributed equally to this work.

  • Sarah A. Kinkel,

    1. Divisions of Molecular Medicine and Immunology, Walter and Eliza Hall Institute of Medical Research Parkville, VIC, Australia
    2. The Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia
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    • These authors contributed equally to this study.

  • François-Xavier Hubert,

    1. Divisions of Molecular Medicine and Immunology, Walter and Eliza Hall Institute of Medical Research Parkville, VIC, Australia
    2. The Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia
    Current affiliation:
    1. Institut National de la Santé et de la Recherche Médicale Unité 643, Centre Hospitalier Universitaire de Nantes, Nantes, France, Institut de Transplantation et de Recherche en Transplantation-Urologie-Néphrologie, Université de Nantes, Nantes, France
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    • These authors contributed equally to this study.

  • Zeyad Nasa,

    1. Department of Immunology, Central Clinical School, Monash University, Melbourne, VIC, Australia
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  • James Chan,

    1. Centre for Inflammatory Diseases, Department of Medicine, Southern Clinical School, Monash University, Clayton, VIC, Australia
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  • Christopher Siatskas,

    1. Monash Immunology and Stem Cell Laboratories, Monash University, Melbourne, VIC, Australia
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  • Premila Hirubalan,

    1. Centre for Inflammatory Diseases, Department of Medicine, Southern Clinical School, Monash University, Clayton, VIC, Australia
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  • Ban-Hock Toh,

    1. Centre for Inflammatory Diseases, Department of Medicine, Southern Clinical School, Monash University, Clayton, VIC, Australia
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  • Hamish S. Scott,

    1. Department of Molecular Pathology, The Centre for Cancer Biology, The Institute of Medical and Veterinary Science and The Hanson Institute, SA Pathology, Adelaide, SA, Australia
    2. The Adelaide Cancer Research Institute, The School of Medicine University of Adelaide, SA, Australia
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    • The laboratories of these authors contributed equally to this work.

  • Frank Alderuccio

    Corresponding author
    1. Department of Immunology, Central Clinical School, Monash University, Melbourne, VIC, Australia
    • Department of Immunology, Monash University, Prahran, 3181, VIC., Melbourne Fax: +61399030018
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    • The laboratories of these authors contributed equally to this work.


Abstract

The autoimmune regulator (AIRE) promotes “promiscuous” expression of tissue-restricted antigens (TRA) in thymic medullary epithelial cells to facilitate thymic deletion of autoreactive T-cells. Here, we show that AIRE-deficient mice showed an earlier development of myelin oligonucleotide glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). To determine the outcome of ectopic Aire expression, we used a retroviral transduction system to over-express Aire in vitro, in cell lines and in bone marrow (BM). In the cell lines that included those of thymic medullary and dendritic cell origin, ectopically expressed Aire variably promoted expression of TRA including Mog and Ins2 (proII) autoantigens associated, respectively, with the autoimmune diseases multiple sclerosis and type 1 diabetes. BM chimeras generated from BM transduced with a retrovirus encoding Aire displayed elevated levels of Mog and Ins2 expression in thymus and spleen. Following induction of EAE with MOG35–55, transplanted mice displayed significant delay in the onset of EAE compared with control mice. To our knowledge, this is the first example showing that in vivo ectopic expression of AIRE can modulate TRA expression and alter autoimmune disease development.

Introduction

In humans, deficiency of the autoimmune regulator (AIRE) results in the autosomal recessive disorder, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy otherwise known as autoimmune polyglandular syndrome type 1 1, 2. Studies of Aire−/− mice confirm AIRE as a transcriptional regulator that controls the intra-thymic expression of peripheral tissue-restricted antigens (TRA) implicated in the induction of immunological tolerance 3–5. While the exact number of genes regulated by AIRE is not known, estimates in mouse and man suggest this may be hundreds to thousands of genes 4, 6–8. Within the thymus, the AIRE protein is expressed predominantly within medullary thymic epithelial cells (mTEC), although expression has also been reported in dendritic cells (DC) 9–11. More recent reports also suggest Aire expression in peripheral cells and tissues 12–15, but its presence and function in these cells still remains an area of debate 9, 16.

The generation of AIRE-deficient mice (Aire−/−) has been instrumental in deciphering the role of AIRE in immune tolerance and susceptibility to autoimmune disease. To date, four Aire−/− mouse models have been reported 4, 17–19 and while there is intra- and inter-strain variation, Aire−/− mice develop a range of organ-specific autoimmune diseases that are generally directed towards specific TRA 4, 17, 20, 21. Furthermore, manipulation of Aire expression can influence central tolerance by altering TRA levels in mTRA and thus predisposition to autoimmunity 22. In human type 1 diabetes mellitus and Myasthenia gravis, a similar scenario may exist where genetic polymorphisms in the regulatory regions of target autoantigen genes INS2 and AChR, respectively, indirectly influences the thymic transcription of these TRA by AIRE 23, 24. Therefore, variations in the level of autoantigen displayed can set the threshold for self-tolerance and co-determine disease susceptibility.

Our interest in autoimmunity focuses on the concept that the ectopic expression of target autoantigens can be used as a means of promoting immune tolerance. In particular, our strategy involves genetic manipulation and transfer of BM cells to provide a source of ectopically expressing cells 25. This process has been shown in numerous studies to promote antigen specific tolerance 26–28. Using the MOG35–55 model of EAE, we have shown that the transplantation of BM cells transduced with a retrovirus encoding myelin oligonucleotide glycoprotein (Mog) can prevent the induction of EAE 29. One potential mechanism that underlies this tolerogenic effect involves the deletion of autoreactive cells in the thymus 29. However, the effectiveness of this approach is potentially limiting given that autoimmune diseases are often associated with epitope spreading, resulting in multiple autoantigens being generated. Since AIRE is known to control the expression of many TRA, we asked whether ectopic expression of AIRE in BM derived cells can promote expression of known autoantigens and whether this can influence the development of EAE.

Studies in which AIRE has been over-expressed in tissue culture cell lines have reported up- and down-regulation of a range of transcripts associated with diverse cellular functions such as adhesion, cell cycle, cytokine signaling, transcription factors, signal transduction and apoptosis, as well as a limited number of TRA 30–33. Transgenic mice, where AIRE is delimited within pancreatic islet beta cells, resulted in the expression of a large array of transcripts not normally found in this tissue 34. However, to date, there are no studies to exploit the TRA promoting properties of AIRE in vivo and address whether ectopic expression of AIRE can influence the development of autoimmune disease.

Results

Ectopic expression of AIRE upregulates autoantigen expression in cultured cell lines

We examined the potential of AIRE to influence TRA expression in cultured cell lines by retroviral transduction with Aire. The cell lines included those derived from thymic epithelium (B6TEA and 427.1), dendritic cells (DC2.5), macrophages (J774 and RAW) and NIH/3T3 fibroblasts. To perform our studies, we generated retroviral vectors that encoded murine Aire (pAire) and as controls, Mog (pMog) or Ins2 (pProII). All constructs also contained a GFP cassette for identification of transduced cells or progeny (Fig. 1A).

Figure 1.

Transduction of in vitro-cultured cell lines with a retroviral vector encoding Aire. (A) Schematic of retroviral vector used to generate retrovirus encoding Aire, Mog or Ins2 (ProII) genes cloned into multiple cloning site (mcs). Expression of enhanced green fluorescent protein (eGFP) was used to mark transduced and progeny cells and controlled by an internal ribosomal entry site (IRES). (B) Thymic epithelial (B6TEA, 427.1), macrophage (J774, RAW2674.4) and NIH/3T3 cells were transduced with an Aire retroviral vector and stained for AIRE with the 5H12 monoclonal antibody (green). Nuclei (Nu) were visualised with Hoechst 33342 (blue). Images were captured by confocal microscopy and merged for colocalisation analysis. Non-transduced and Aire-transduced cells are indicated. Scale bar represents 50 μm. Higher magnification images (×400) of boxed region within merged image of Aire-transduced cells are shown to illustrate staining pattern of retroviral-driven AIRE expression. Scale bar represents 15 μm. (C) Expression of AIRE protein in B6TEA and 427.1 thymic epithelial cell lines by flow cytometry in non-transduced and retroviral-transduced cells.

Cells were transduced with pAire and transduced cells identified by the expression of GFP. To confirm AIRE protein expression, transduced cells were stained with a monoclonal antibody specific to the AIRE protein 9. Confirming previous reports 9, 35, fluorescence microscopy revealed a punctate staining pattern what was localised within the nucleus. Co-localisation of AIRE with cytoskeletal filaments was also observed in some cells as previously been reported in Aire-transfected cell lines 36–38. All non-transduced cell lines failed to stain for AIRE, suggesting that the endogenous AIRE expression was lacking or at undetectable levels (Fig. 1B). AIRE expression, as assessed by flow cytometry was maintained in GFP+ cells even after several passages in cell culture (Fig. 1C). GFP+ cells continued to grow well in culture without any obvious adverse effect on doubling time or survival.

Having established this panel of AIRE-expressing cell lines, we asked whether AIRE expression was sufficient to activate the expression of a panel of TRA; thus, potentially mimicking the role of AIRE in the thymus. The TRA selected for quantitative RT-PCR (qRT-PCR) represented autoantigens associated with defined autoimmune diseases such as type 1 diabetes (Ins2), EAE/MS (Mbp, Mog, Plp1), autoimmune gastritis (Atp4a), hypothyroidism (Nalp5), uveitis (Rbp3) and Sjögren's syndrome (Spt1). Spna2 (α-fodrin) was included as a negative control, and although identified as a target autoantigen in Sjögren's syndrome-like pathology in Aire−/− mice, its expression in the thymus is independent of AIRE 18. Corroborating immunofluorescence studies, Aire transcript levels in transduced cell lines were at least 10 000-fold above non-transduced cells (Fig. 2A). As predicted, Spna2 expression was unaltered across the cell lines.

Figure 2.

Expression of Aire and TRA in transduced in vitro-cultured cells. (A) RNA isolated from non-transduced cells and pAire-transduced cell lines was subjected to qRT-PCR. The quantity of each target gene was normalised against housekeeping gene Hprt and expression levels represented for transduced cells are relative to non-transduced cells. Each data point represents the mean of three replicates and each cell line was independently tested twice or three times. (B) Non-transduced cells (427.1), and cells transduced with retrovirus encoding Aire (427.1-Aire) or Mog (NIH/3T3-MOG) were grown on glass chamber slides and stained with MOG-specific monoclonal antibody 8–18C5. Co-localisation of GFP+ and MOG is shown in merged panels.

We observed that the level of TRA mRNA modulation was not consistent across the different cell lines. The transduced thymic derived cell lines (B6TEA and 427.1) expressed a greater number of TRA in comparison with other cell lines tested; however, the expression of specific TRA differed between these lines (Fig. 2A). For example, Mog was highly upregulated in transduced 427.1 cells but was unaltered in B6TEA cells, whereas the expression of another myelin antigen gene, Mbp, was upregulated in B6TEA, but was unaffected in 427.1 cells. Further highlighting this heterogeneity was the observation that Atp4a displayed higher expression in B6TEA and 427.1 thymic epithelial cell lines compared with the macrophage (J774 and RAW267.4) and fibroblast lines (Fig. 2A). Given the relatively high expression of Mog we observed in 427.1 cells we examined these cells for MOG protein expression using an anti-MOG specific monoclonal antibody 29. Aire-transduced cells expressing GFP (and thus AIRE) were specifically reactive with the anti-Mog monoclonal antibody, confirming that the expression of AIRE in these cells promotes MOG expression (Fig. 2B). Non-transduced 427.1 did not display any MOG reactivity, and staining of control cells (NIH/3T3) transduced with retrovirus encoding Mog demonstrates the specificity of the anti-Mog monoclonal antibody in transduced (GFP+) cells (Fig. 2B). These data confirm that ectopic expression of AIRE in cultured cell lines can influence expression of TRA in a cell context-dependent manner and that Aire-transduced cells can express detectable levels of MOG protein.

Transplantation of Aire-transduced BM cells upregulates MOG expression in chimeric mice

We have recently shown that the transplantation of BM transduced with pMog promotes deletional tolerance and prevents development of the MOG35–55-induced EAE in C57BL/6 mice 29. Given that the ectopic expression of AIRE can induce expression of TRA, including MOG in vitro, we asked whether the transplantation of retrovirally transduced BM cells expressing AIRE in syngeneic animals altered the course of EAE in animals immunized with MOG35–55. The level of chimerism was analysed 10 weeks following the transplantation of transduced BM cells by assessing the percentage of GFP+ cells from the thymus and spleen. The GFP expression was detected in all the major cell lineages examined, including CD4+and CD8+ T cells, B cells and MHC class II+ CD11c+ dendritic cells (Fig. 3A, Supporting Information Fig. 1 for gating strategy). RT-PCR analysis of thymus samples from Aire chimeric mice revealed increased levels of Aire, Mog and Ins2 mRNA compared with thymi from mice transplanted with normal BM or from untouched WT mice, suggesting that the AIRE expression has upmodulated these two defined autoantigens (Fig. 3B). While attempted, we were not able to accurately quantify and compare the MOG expression in the thymus across normal mice, mice transplanted with normal BM or Aire-transduced BM. To demonstrate differential expression of Aire and TRA in cells originating from transduced BM cells, GFP+ cells were enriched from the spleens of chimeric mice. Comparison of GFP+ and GFP- cells indicated a greater level of AIRE expression in GFP+ cells, consistent with retroviral promoter-driven expression within these cells. Further analysis revealed elevated levels of Mog and Ins2 mRNA in GFP+ cells compared with GFP- cells (Fig. 3C). These data support our in vitro findings that the ectopic expression of AIRE can promote the expression of TRA including the autoantigens Mog and Ins2.

Figure 3.

Aire chimeric mice express elevated levels of Mog in the thymus and spleen. BM stem cells transduced with retrovirus encoding Aire, Mog, Ins2 (ProII) or left non-transduced were transplanted into irradiated mice and analysed at 10 weeks for chimerism and Aire and autoantigen expression. (A) Single cell suspensions from thymus and spleen were assessed by flow cytometry for GFP expression in indicated immune cell populations. Group sizes are indicated and data show mean±SEM. (B) RNA was isolated from thymi of WT C57BL/6 mice and mice receiving normal or Aire transduced BM and subjected to RT-PCR to detect expression of Aire, Mog and ProII. Expression of housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as loading control. Images were captured with digital camera and have been everted. (C) GFP+ and GFP- cells were purified by flow cytometry from the spleens of chimeric mice transplanted with Aire-transduced BM and subjected to RT-PCR analysis of Aire, Mog and ProII mRNA expression. Expression of Gapdh was used as loading control. Images were captured with digital camera.

Aire–/– mice have lower MOG expression in mTEC and are more susceptible to equation image-induced EAE

We next determined whether the intrathymic expression of the EAE/MS associated autoantigens Mog, Plp and Mbp was AIRE dependent. MHCIIhi mTEC (CD45, Ly51, MHCIIhi) from WT and Aire−/− C57BL/6 mice were isolated and qRT-PCR revealed a marked reduction in the expression of MOG (to 25% WT levels) and Plp (to 12% WT levels) in Aire−/− mTEC with no change in Mbp expression (Fig. 4A). While Mog has previously been reported as being AIRE dependent, PLP was reported to be AIRE independent 39. However, these data came from human association studies of AIRE and TRA expression rather than from the examination of AIRE-deficient thymi and could thus explain the discrepancy in result for PLP. Given the observed reduction in Mog expression, we asked whether Aire−/− mice were more susceptible to MOG-induced EAE than WT C57BL/6 mice. We found that Aire−/− mice immunised with MOG35–55 developed EAE significantly earlier than WT mice (p=0.007) (Fig. 4B). Histological analysis did not reveal any differences in CNS pathology between knockout and control groups with severe mononuclear cell infiltrate and axonal demyelination in CNS lesions in both groups (not shown). These studies suggest that Mog expression is regulated by AIRE and this can influence the development of MOG35–55-induced EAE.

Figure 4.

Induction of EAE in Aire−/− mice and chimeric mice ectopically expressing AIRE. (A) qRT-PCR was performed on MHCIIhi mTEC from WT and Aire−/− mice to assess expression levels of Aire and TRA Mog, Mbp and Plp1. Expression levels were normalised to housekeeping gene Hprt and relative expression is shown. (B) C57BL/6 WT and Aire−/− mice were immunised with 200 μg MOG35–55 peptide and pertussis toxin and scored for the development of EAE. Group sizes for each experiment are indicated. Data show mean±SEM and significance between curves evaluated by permutation test as detailed in Material and methods section. (C) WT C57BL/6 mice (EAE induction control), or chimeric mice transplanted with normal bone marrow (nBMT) or BM transduced with retrovirus pAire, pMog or pProII were immunised with MOG35–55 peptide in CFA and pertussis toxin. Mice were monitored daily and scored for development of EAE. Group sizes are indicated and represent the data combined from two independent experiments. Data show mean±SEM. Statistical analysis between groups was performed as detailed in the Materials and methods section and p-values are presented in tabular form.

Transfer of BM cells transduced with Aire attenuates development of MOG-induced EAE

As a therapeutic strategy that is in line with our previous studies 29, we asked whether AIRE-induced MOG expression in chimeric mice following transplantation of Aire transduced BM would prevent or reduce the development of EAE. Cohorts of lethally irradiated C57BL/6 mice were transplanted with non-manipulated BM cells or BM cells transduced with either pAire, pProII or pMog retrovirus. Ten weeks after transplantation, mice were immunised with MOG35–55 peptide and monitored for EAE development. Chimeric mice ectopically expressing AIRE had a significantly delayed initiation and progression of EAE compared to control groups (Aire versus ProII, p=0.009; versus nBMT, p=0.002; versus WT control, p=0.001) (Fig. 4C). There was no difference between the control groups (all p values>0.2) except for the positive control group that ectopically expressed MOG directly and did not develop EAE (Aire versus Mog, p=0.002). The absence of EAE in MOG chimeric mice confirms our published data that mice transplanted with Mog-transduced BM are resistant to EAE induction 29. These observations suggest that ectopic expression of AIRE promotes elevated levels of MOG expression in BM derived cells and that this can delay the development of EAE following MOG35–55 immunisation.

Discussion

The ability to genetically manipulate the BM compartment and promote ectopic antigen expression and immune tolerance has been demonstrated in a number of settings 26–28, 40. We have recently shown that transduced BM cells encoding Mog led to ectopic expression of MOG in BM-derived cells and immune tolerance with complete resistance to EAE induction 29. It is well established that the transcription factor AIRE is associated with the expression of a large array of TRA in the thymus 4, 5 and to a lesser extent in the periphery 13. Furthermore, mice and humans lacking AIRE have a greater incidence of autoimmune conditions 4, 17–19. We therefore asked whether the ectopic expression of AIRE could be used to promote the ectopic expression of target autoantigens and whether this could influence the susceptibility to MOG-induced EAE.

While AIRE expression in vivo is predominantly restricted to thymic medullary cells, it has also been detected outside the thymus in dendritic cells and peripheral lymphoid organs 13, 16. Following the in vitro transduction of a number of cell lines of thymic, dendritic cell and macrophage origin with an Aire-encoding retrovirus, we observed that indeed the expression of TRA was upregulated in an AIRE-dependent manner. Our observation that the TRA expression was not uniform and could vary across cell types is not dissimilar to a recent study examining the effect of AIRE in transgenic mice where AIRE expression was targeted to the pancreatic islet β cells 34. While the AIRE expression in β cells did induce TRA expression, when compared with thymic medullary epithelial cells, the authors found minor overlap in the gene expression patterns. This suggests a cell specific aspect to the expressed AIRE and that AIRE has the general ability to promote the TRA expression regardless of where it may be expressed 34.

Prompted by our in vitro observations, we generated a panel of chimeric mice to test whether the ectopic expression of AIRE through transfer of transduced BM can influence the development of EAE. As previously published, we confirmed that the ectopic expression of MOG following transplantation of BM transduced by retrovirus encoding Mog prevented EAE development 29. While transplantation of Aire-transduced BM did not completely protect mice from EAE development, there was significant retardation in the induction of EAE compared with control groups. In our earlier studies with ectopic expression of MOG, we observed evidence of thymic deletion of MOG35–55-specific T cells 29. We predict that a similar mechanism may also be active here but this needs to be confirmed. While the ectopic gene expression in our system is not restricted to any particular cell lineage due to the ubiquitous nature of the retroviral promoter, dendritic cells would be considered the main BM-derived instigator of tolerance 41, 42 through uptake and presentation of antigen 43, 44. However, it has been shown that if dendritic cells can directly express antigen, then tolerance to that antigen can also ensue 45. Given this, we suggest that MOG expressed within dendritic cells derived from transduced BM could drive tolerance within the thymus through deletion and/or possibility through the generation of T regulatory cells 46. Our model will also promote the ectopic AIRE expression in the range of peripherally destined cells such as dendritic cells, macrophages and B cells, and thus cannot be overlooked at this stage as another potential avenue for mechanisms capable of promoting tolerance. Finally, we cannot rule out the possibility that the ectopic expression of Aire may be exerting its effect on EAE independently of TRA expression. AIRE is also known to transcriptionally activate or repress non-TRA, such as cytokine and cytokine receptors 47 and thus could influence immune responses. Whether a similar effect is occurring in our model of ectopically expressed Aire is not known at this point.

Autoimmune diseases remain a major clinical challenge and current treatments are non-curative and often involve non-specific immunosuppressive regimes. The prospect of developing strategies aimed at delivering antigen-specific tolerance would be a major advance in this field. The ability to influence the immune system through genetic manipulation of the BM compartment offers one avenue for developing novel therapeutic approaches 25. Antigen-specific tolerance driven by transduction of haematopoietic stem cells has now been demonstrated for a range of targets including neoantigens 26, alloantigens 40, allergens 27 and autoantigens 28, 29, demonstrating the feasibility of this approach. In this study, we have exploited the knowledge that AIRE is associated with the expression of TRA in the thymus to demonstrate that it will also promote TRA expression in novel environments. We have demonstrated in the mouse model of EAE that the chimeric mice generated through transduction of BM with Aire ectopically express Mog and are more resistant to MOG35–55-induced EAE induction than WT mice. In summary, our studies have demonstrated the possibility of utilising Aire to treat autoimmune diseases with broad autoantigenic profiles.

Materials and methods

Mice

Female C57BL/6 mice were obtained from Monash Animal Services (MAS, Australia). BM donors were 5–6 weeks old, whereas BM recipients were 6- to 10-week-old mice. Animals were housed in specific pathogen-fee conditions (Monash Medical Centre Animal Facilities MMCAF Australia). Aire−/− mice have been previously described 17. All experiments were performed in accordance with local animal ethics committee approval.

equation image-induced EAE

EAE was induced by subcutaneous injections (femoral regions) of 200 μg MOG35–55 peptide (GL Biochem, Shanghai, China) emulsified in CFA (Sigma) and supplemented with 4 mg/mL Mycobacterium tuberculosis. Mice also received 350 ng pertussis toxin (Sigma-Aldrich) intravenously at time of immmunisation and 48 h later. Animals were monitored daily. Neurological impairment was scored on an arbitrary clinical score: 0, no clinical sign; 1, limp tail; 2, limp tail and hind limb weakness; 3, severe hind limb paresis; 4, complete hind limb paresis; 5, moribund or death. At the completion of the experiment, the brain and spinal cord was taken for histological analysis.

Retroviral constructs

Mouse Aire cDNA 48 was subcloned into retroviral vector pMYs-IRES-eGFP 49 to generate the pMYs-Aire-IRES-eGFP vector encoding Aire (pAire). Retroviral vectors encoding mouse Mog, pMYs-MOG-IG (pMOG) and proinsulin II (Ins2), pMYs-ProII-IG (pProII) have previously been described 29, 50. Recombinant retroviruses were generated using the BOSC23 producer cell line or co-transfection of 293T cells with pPAM-E and pVSVG. Viral titres were determined on NIH3T3 cells 50.

In vitro-cultured cell lines

Thymic epithelial cell lines B6TEA and 427.1, macrophage lines J774 and RAW2674.4, dendritic cell line DC2.4 and NIH3T3 fibroblasts were cultured in DMEM supplemented with 10% FBS, L-glutamine, penicillin and streptomycin. Cell lines were transduced with retroviral supernatant and eGFP+ cells sorted by flow cytometry for continued culturing and experimental studies.

BM isolation and transduction

Donor mice were treated with 5-fluorouracil (150 mg/kg body weight) 3.5 days before BM harvest. BM cells were cultured in DMEM/10% FBS supplemented with recombinant cytokines: rmIL-6 (10 ng/mL, R&D Systems) and rmSCF (50 ng/mL, R&D Systems). After 24 h, cells were transduced with retroviral supernatant by spin-infection 49 and cultured for a further 3–4 days before transferring sorted eGFP+ BM cells into recipient mice preconditioned with 2×550 cGy total body irradiation. Between 20 000 and 200 000 eGFP+ cells were transferred via tail intravenous injections. One day later, radioresistant host T cells were depleted by treatment of BM recipients and untreated control groups with anti-Th1.1 (clone T24) antibody. Mice were left to reconstitute for 8–10 weeks before immunisation. Levels of chimerism were determined 5 weeks post BMT through blood analysis and extensively at completion of experiment.

Isolation of mTEC

mTEC were enriched from thymus as described by Gray et al. 51. Thymi from 10–12 adult mice (6–10 weeks old) were collected in MT-RPMI. After the removal of excess fat and connective tissue, small cuts were made around the edges of the thymic lobes. Following a brief agitation using a wide bore glass pipette, the sample was then subjected to enzymatic digestion. Thymic fragments were incubated in 5 mL of 0.125% w/v collagenase D with 0.1% w/v DNAse I (Roche) in MT-RPMI at 37°C for 15 min. Cells released into suspension were removed after larger thymic fragments had settled and fresh enzyme containing media was added to the intact thymic lobes. This was repeated 3–4 times with fresh media. In the final digest, collagenase D was replaced with trypsin (Roche) and incubation time was extended to allow for complete digestion of thymi lobules.

Each fraction was counted and the final 2 or 3 enrichments, which contained a higher proportion of CD45 cells, were pooled to obtain 100×106 total cells. A negative depletion was performed to enrich for CD45 cells using CD45 microbeads (Miltenyi Biotec) and the AutoMACS system (Miltenyi Biotec), using the DepleteS program. The CD45 cell fraction was then resuspended in KDS-BSS with 3% v/v FBS and stained using the following antibodies: anti-CD45-APC (30F11; BD Biosciences), anti-MHCII-PE (M5/114.15.2; BD Biosciences) and anti-Ly51-FITC (6C3; BD Biosciences). Prior to sorting, 0.5 μg/mL PI (Calbiochem) was added to each samples to allow for the exclusion of dead cells. Cells were sorted using the FACSAria (BD Biosciences).

RT and Quantitative RT-PCR (RT-PCR and qRT-PCR)

RNA from cultured cells, whole tissues or sorted cells was prepared using the RNeasy Mini-kit (Qiagen) including an on-column DNaseI digest as per manufacturer's protocol. cDNA was generated using Superscript III RT (Invitrogen) as per manufacturer's protocol. For RT-PCR the primers used were: Aire; For 5′-accatggcagcttctgtccag-3′, Rev 5′-gcagcaggagcatctccagag-3′; Ins2; For 5′-accatcagcaagcaggaag-3′, Rev 5′-ctggtgcagcactgatctacaatgc-3′; Mog; For 5′-ggactagtgactctgtccccggtaaccat-3′, Rev 5′-ggactagtctcgagagaaccatcactcaaaagggg-3′, Gapdh; For 5′-catgacaactttggcattgtgg-3′, Rev 5′-cagatccacaacggatacattggc-3′. PCR conditions were optimized for each primer set. Annealing temperature and number of cycles were modified from standard thermal cycler conditions as follows: a single denaturing step at 94°C for 2 min, followed by 25 cycles (for Aire and Gapdh) or 35 cycles (for Mog) of 92°C for 30 s, 60°C for 30 s and 72°C for 2 min, followed by a final extension step of 72°C for 5 min. Ins2 was amplified for 35 cycles with an annealing temperature of 65°C. PCR products were analysed by 1% agarose gel electrophoresis containing 0.5 μg/mL of ethidium bromide. Images were captured using a Bio-Rad Gel Doc XR system (Bio-Rad Laboratories).

Quantitative RT-PCR was performed using Roche LightCycler 480 System with the following primers designed using the Universal Probe Library assay design centre: Hprt: For 5′-tcctcctcagaccgctttt-3′, Rev 5′-cctggttcatcatcgctaatc-3′, probe ♯95; Aire; For 5′- tgctagtcacgaccctgttct-3′, Rev 5′- ggatgccgtcaaatgagtg-3′, probe ♯109; Atp4a; For 5′-aatgggaggaccaccatcta-3′, Rev 5′-aggcgctgaccaaatgtc-3′, probe ♯72; Spt1; For 5′-tgctcttctacttgtcaccatga-3′, Rev 5′-tgtttgtctccgggtcct-3′, probe ♯72; Ins2; For 5′-gaagtggaggacccacaagt-3′, Rev 5′-agtgccaaggtctgaaggtc-3′, probe ♯32; Spna2; For 5′-gctagtcactatgcctcagatgaa-3′, Rev 5′-aagctcccacagctccag-3′, probe ♯91; Mog; For 5′-cttcttcagagaccactcttacca-3′, Rev 5′-gttgacccaatagaagggatctt-3′, probe ♯34; Mbp; For 5′-cctcagaggacagtgatgtgttt-3′, Rev 5′-agccgaggtcccattgtt-3′, probe ♯16; Plp1; For 5′-tcagtctattgccttccctagc-3′, Rev 5′-agcattccatgggagaacac-3′, probe ♯53; Rbp3; For 5′-atgactcggtcagcgaactt -3′, Rev 5′-gatggctacgctcttcttgg -3′, Probe ♯100; Nalp5; For 5′-caatgccctgtctctaacctg -3′, Rev 5′-tgtcttctcactcgggcata -3′, Probe ♯38. All qRT-PCR reactions were prepared in 10 μL with final concentrations of 1× LightCycler 480 Probes Master, 200 nM forward and reverse primers, and 100 nM Universal ProbeLibrary probe (Roche Applied Science), using the following cycling conditions: 95°C for 10 min, followed by 45 cycles of 95°C for 10 s and 60°C for 30 s, followed by 40°C 1 min to cool. Crossing-point (Cp) values were calculated using the second derivative maximum method performed by the LightCycler 480 quantification software (Roche Applied Science). Serially diluted cDNA was used to construct a four-point standard curve for each qRT-PCR assay. The starting quantity (arbitrary units) of cDNA for each gene was then calculated as a linear function of the logarithmic concentration and Cp. The starting quantity of each target gene was normalised to the starting quantity of housekeeping gene Hprt for each sample. Expression is shown relative to non-transduced cell lines.

Flow cytometry

Single cell suspensions from thymus, spleen lymph nodes and BM were prepared by gently dissociating tissues between the frosted ends of glass slides. Tissue cultured cells were collected by trypsin digest for adherent cells lines or collection of culture media. Cells were washed and resuspended in PBS for staining. Monoclonal antibodies (BD Pharminogen) used to stain the following cell surface markers were; CD4 (clone RM4-5), CD8 (clone 53–6.7), CD19 (clone 1D3), CD11c (clone HL3), MHC-II (clone AF6-120.1), B220 (clone RA3-6B2). Intracellular AIRE staining was performed using the BD Cytofix/Cytoperm kit according to the manufacturer's instructions 9. Cell sorting and analysis were performed on FACS (DakoCytomation MoFlo®, DakoCytomation MoFlo® XDP, BD FACSAria™, BD FACSCanto™, BD FACSCalibur™).

Indirect immunofluorescence

Normal and transduced cells were plated on chamber slides (ICN Biomedicals) and permeabilised using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit. For AIRE staining, cells were incubated with monoclonal rat anti-AIRE Ab (Clone 5H12) followed by Alexa 568 nm goat anti-rat IgG (H+L) (Invitrogen). For the detection of MOG protein, cells were stained with monoclonal mouse anti-MOG Ab (Clone 8-18C5; gift from Prof. C Bernard, MISCL, Monash University, Victoria, Australia) followed by secondary Ab (Alexa 594 nm goat anti-mouse IgG). Slides were mounted using Dako Fluorescence mounting medium (Dako Cytomation) and images acquired with an Olympus IX71 Inverted Research Microscope. For confocal microscopy, transduced cells were cultured on glass coverslips, fixed with 4% PFA in PBS and permeabilised with 1% Triton X-100 in PBS prior to staining. Cells were stained with FITC-conjugated anti-AIRE 5H12 9 and nuclear stain Hoechst 33342 (Sigma), mounted using fluorescent mounting media (Dako) and images acquired on a confocal microscope (Leica TCS SP2, Leica Microsystems).

Statistical analysis

Statistical significance was evaluated using two-tailed Student's t test for 2 groups. p values less than or equal to 0.05 were considered significant (*p≤0.05, **p≤0.01, ***p≤0.001). Significant difference between two curves was evaluated via a permutation test offered by the Walter and Eliza Hall Institute for Medical Research (Melbourne, Australia) (http://bioinf.wehi.edu.au).

Acknowledgements

We thank K. Webster for help with mTEC isolation and P. Crewther for animal and laboratory management. We thank AMREP and WEHI Animal Services for animal care and management. This work was supported by fellowships from La Fondation pour la Recherche Medicale (FRM) and the 6th FP of the EU, Marie Curie, contract 040998 (to F.-X.H.), by Australian Postgraduate Awards (to S. A. K), NHMRC fellowships (171601 and 461204), NHMRC program grants (257501, 264573, 406700), Eurothymaide and EURAPS, 6th FP of the EU, and the Nossal Leadership Award from the Walter & Eliza Hall Institute of Medical Research to H. S. S., and NHMRC project grant (491004), to F. A., H. S. S. and F. X. H.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

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