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Keywords:

  • Experimental autoimmune encephalomyelitis;
  • gene therapy;
  • hematopoietic stem cell transfer;
  • multiple sclerosis;
  • nonmyeloablative

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Autoimmune diseases result from chronic targeted immune responses that lead to tissue pathology and disease. The potential of autologous hematopoietic stem cells transplantation as a treatment for autoimmunity is currently being trialled but disease relapse is an issue. We have previously shown in a mouse model of experimental autoimmune encephalomyelitis (EAE) that the transplantation of bone marrow (BM) transduced to encode the autoantigen myelin oligodendrocyte glycoprotein (MOG) can prevent disease induction. However these studies were performed using lethal irradiation to generate BM chimeras and a critical factor for translation to humans would be the ability to utilize low toxic preconditioning regimes. In this study, treosulfan was used as a nonmyeloablative agent to generate BM chimeras encoding MOG and assessed in models of EAE induction and reversal. We find that treosulfan conditioning can promote a low degree of chimerism that is sufficient to promote antigen specific tolerance and protect mice from EAE. When incorporated into a curative protocol for treating mice with established EAE, nonmyeloablative conditioning and low chimerism was equally efficient in maintaining disease resistance. These studies further underpin the potential and feasibility of utilizing a gene therapy approach to treat autoimmune disease.


Abbreviations: 
BM

bone marrow

BMT

bone marrow transfer

BSA

bovine serum albumin

EAE

experimental autoimmune encephalomyelitis

FCS

fetal calf serum

GFP

green fluorescent protein

HSC

hematopoietic stem cells

HSCT

hematopoietic stem cell transfer

IP

intraperitoneal

IRES

internal ribosomal entry site

MOG

myelin oligodendrocyte glycoprotein

PBS

phosphate buffered saline

ProII

proinsulinII

rmSCF

recombinant mouse stem cell factor

rmIL-6

recombinant mouse interleukin 6

SEM

standard error of the mean

TBI

total body irradiation

Treo

treosulfan

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Autoimmune diseases affect 5–6% of the population and represent antigen-specific immune responses that result in a range of clinical syndromes (1,2), with no known cures. Autologous hematopoietic stem cell transfer (HSCT) as a treatment for human autoimmune disease is currently being explored and involves the reintroduction of collected hematopoietic stem cells (HSC) into preconditioning recipients, in a process aimed at depleting pathogenic clones and ‘resetting’ the hematopoietic system (3,4). Outcomes in individuals across a range of autoimmune diseases, including multiple sclerosis have been beneficial (3,5–9), yet relapses are frequent and presumably due to reemerging or transfer of self-reactive clones. Because there is nothing inherent in the process of HSCT to drive immunological tolerance, we have incorporated the notion of ectopic antigen expression to promote antigen specific tolerance (10). Early studies in transgenic mouse models demonstrated that widespread ectopic expression of autoantigen resulted in antigen specific resistance in a range of disease models (11–13). More recently, we and others have demonstrated that transplantation of BM cells transduced with retrovirus encoding a range of antigens can promote antigen specific tolerance and disease resistance (14–18).

Translation of such a strategy to humans will require demonstration of clinical feasibility. These early studies were performed using lethal total body irradiation (TBI) to demonstrate the concept that a gene therapy approach can be used to generate tolerance and prevent autoimmune diseases. We have since shown that tolerance and resistance to EAE can be achieve with nonlethal doses of irradiation (19) but preconditioning with TBI is not the choice for HSCT in human autoimmune disease and a key aspect of current protocols involving HSCT is to reduce preconditioning associated events (6,20,21). Therefore it remains to be demonstrated in a defined model of autoimmunity that the use of a clinically relevant nonmyelobalative approach can successfully be used to promote chimerism, immune tolerance and disease resistance.

In this study, we have extended our current knowledge to demonstrate that under nonmyeloablative chemotherapeutic conditioning, the transfer of BM transduced with retrovirus encoding the autoantigen MOG can generate chimerism in hematopoietic cells, immune tolerance to MOG and resistance to EAE induction. Our studies demonstrate that not only are minimal levels of chimerism required to achieve tolerance and disease resistance, but this strategy is equally effective in treating established EAE. Overall, these studies provide evidence that gene therapy can be used in a clinically relevant scenario to promote immunological tolerance in the context of treating autoimmune disease.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Mice

C57BL/6 female mice were obtained from and housed within the Alfred Medical Research and Educational Precinct animal center. All experiments were performed with approval from the institutional animal ethics committee.

Induction of EAE and clinical scoring

EAE was induced by immunization with MOG35–55 peptide as previously described (15). Neurological impairment was scored on an arbitrary scale of 0–5 as follows. 0: no clinical signs; 1: limp tail; 2: limp tail and hind limb weakness; 3: severe hind limb paresis; 4: forelimb weakness and hind limb paralysis; 5: moribund or death. Mice demonstrating a score of 3 or above were killed in accordance with ethics requirements.

Treosulfan treatment

Treosulfan doses were based on the published studies of Van Pel (22,23). Treosulfan (L-threitol-1,4-dimethanesulfonate, Medac, Germany) was resuspended in sterile water at a concentration of 50 mg/mL and administered by IP injection over 3 consecutive days. In experiments designed to determine optimal timing for BMT, mice received a dose of 6000 mg/kg and transferred with BM 48, 72 or 96 h after the final injection. In studies comparing myeloablative (6000 mg/kg) and nonmyeloablative (4500, 3000 or 1500 mg/kg) doses, mice were transferred with BM 72 h after the final injection.

Methylprednisolone-induced remission of EAE

Methylprednisolone (Sigma-Aldrich, USA) was used to induce remission in C57BL/6 mice with MOG35–55 induced EAE as previously described (24). Mice were first immunized with MOG35–55 peptide as described above and upon reaching a clinical score of 2, treated with methylprednisolone (10 mg/kg/day) in the drinking water to promote remission (24).

Retroviral vectors

MOG cDNA was generated from C57BL/6 mouse brain mRNA and subcloned into retroviral vector pMYs-IG to create pMYs-MOG-IG as previously described (15). Mouse Proinsulin II (ProII) cDNA was subcloned by polymerase chain reaction (PCR) from NOD mouse genomic DNA to generate retroviral vector pMYs-ProII-IG as previously described (25). These retroviral vectors also encoded green fluorescent protein (GFP) driven by an internal ribosomal entry site (IRES) to enable tracking and enumeration. Recombinant retrovirus was generated by transfection of BOSC23 cells (26) with pMYs-MOG-IG and pMYs-ProII-IG and supernatants collected at 48 and 72 h posttransfection. Retroviral titers were determined by transduction of NIH3T3 cells and approximately 1× 107 infectious units (IU)/mL.

Bone marrow harvest, retroviral transduction and transfer

Donor C57BL/6 mice were injected IP with 5-fluorouracil (Hospira, Australia) at 150 mg/kg, 4 days before BM harvest. Bone marrow was flushed from femurs and tibias with 1 × Hank's balanced salt solution (Invitrogen, Carlsbad, CA, USA) supplemented with 1% penicillin and streptomycin (Invitrogen), filtered through 70 μm strainer and red blood cells lysed with lysis buffer (Sigma Aldrich, St. Louis, MO, USA). Cells were plated into 24-well plates at 2.5–3× 106 cells/well in DMEM (Invitrogen), 10% fetal calf serum (FCS; Invitrogen) supplemented with recombinant mouse cytokine IL-6 (rmIL-6, 10 ng/mL, R&D Systems, Australia) and recombinant mouse stem cell factor (rmSCF, 50 ng/mL, R&D Systems). Twenty four h and 72 h postplating, cells were spin-infected with 2 mL of viral supernatant containing polybrene (5 mg/mL, Sigma-Aldrich) and 10 mM HEPES buffered solution (Sigma-Aldrich) for 1.5 h at 680 G. Viral supernatant was replaced with fresh DMEM/10% FCS, rmIL-6 and rmSCF at doses described above. Cells were cultured for a further 4 days with a change of media and rmIL-6 and rmSCF every 2 days. For BMT, cells were collected, pelleted and resuspended in sterile phosphate buffered saline (PBS). Recipient mice were preconditioning prior to BMT with treosulfan as described, or TBI (2 × 550 cGy). Bulk transduced (nonsorted) cells were injected into the lateral tail vein in a total volume of 100 μL. In the curative study, mice received a single dose of 100 μL antimouse-Thy1.1 (clone T24 supernatant, WEHI monoclonal antibody laboratory, Melbourne, Australia) IP on the day of BMT to deplete circulating T cells.

Enzyme linked immunosorbent assay (ELISA)

Ninety-six-well plates (Corning, Midland, MI, USA) were coated with 100 μL MOG35–55 peptide (5 μg/mL in a carbonate buffer PH 9.6) and incubated over night at 4 °C. Noncoated wells were used to determine background binding. Wells were blocked for 1.5 h at room temperature (RT) with 200 μL of 1% w/v bovine serum albumin (BSA) in PBS. Between steps, wells were washed 4× with washing buffer (1×PBS/0.05% 20–20). Fifty micoliters of mouse sera diluted 1/100 was added in duplicate to MOG35–55 coated and noncoated wells and incubated at RT for 2 h. Plates were washed and 100 μL of horseradish peroxidase conjugated rabbit antimouse IgGγ (Invitrogen) diluted 1/2000 in 1 × PBS/0.05%BSA was added to each well. Wells were incubated for 2 h at RT, washed and incubated in the dark with 100 μL of ABTS (2,2-azino-di(3-ethylbenzthiazoline) sulfonic acid) solution (Invitrogen) per well for 15–30 min. Color reactions were stopped by adding 100 μL of 0.01% sodium azide in 0.1 M citric acid. Optical density for each well was read at 514 nm in a Multiscan®Thermo reader, (Fisher Scientific, Finland) and the final absorbance for each sample calculated by deducting the mean optical density of the noncoated wells from the mean of MOG35–55 coated wells.

Flow cytometry

Single cell suspensions from spleen and thymus were prepared by dissociating tissues through a 70 μm nylon mesh in sterile PBS. Cells (0.2–1×106) were plated in 96-well plates and incubated for 30 min with directly conjugated antibodies, washed with 250 μL PBS/1% FCS, resuspended in 200 μL PBS/1%FCS and analyzed using a LSRII flow cytometer with BD-Diva software (Becton Dickinson, Franklin Lakes, NJ, USA). The following monoclonal antibodies were used: anti-CD4-APC, CD4-pacific blue, anti-CD8-APCCy7, anti-CD11b-PECy7, anti-CD11c-APC, anti-CD19-PECy7, anti-MHC II (IAb)-PE and anti-Gr1 (Ly-6G/C-APC (Becton Dickinson, USA).

Splenocyte proliferation assay

Splenocytes (1 × 106) were cultured in 96-well tissue culture plates in 100 μL RPMI supplemented with 10% FCS, 0.1% 2-mercaptoethanol, 1% L-glutamine, penicillin and streptomycin (Invitrogen) at 37°C, 5% CO2 and stimulated with 1–100 μg/mL MOG35–55 peptide in a total volume of 200 μL. Concanavalin A (2 mg/mL) was used as a positive control. Cells without peptide stimulation were used as background. After 72 h incubation, 10 μL [3H]thymidine (1μCi/well; Amersham) was added to each well and incubated for a further 24 h. Incorporated [3H]thymidine was determined with Packard microplate scintillation and luminescence counter (Packard Biosience). All samples were performed in triplicates and proliferation is presented as a stimulation index.

Histology

Spinal cords were collected and processed for histology as previously described (24) and 5–8 sections were scored blind for inflammatory infiltrate in the meninges or parenchyma and presented as previously described (27).

Quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR)

Total mRNA from splenocytes was prepared using an RNeasy RNA isolation kit (QIAGEN, Germany), treated with DNase I (QIAGEN, Germany) and reverse transcribed using the Superscript™ III First-Strand Synthesis System for RT-PCR with oligo dT (Invitrogen). Quantitative RT-PCR was performed using the ABI prism 7900HT SDS System (Applied Biosystems, Foster City, CA, USA) with primers designed using the Universal Probe Library assay design center. Primer sets for MOG were sense, 5′-CTTCTTCAGAGACCACTCTTACCA-3′; antisense: 5′-GTTGACCCAATAGAAGGGATCTT-3′ and for house keeping gene; mouse hypoxanthine phosphoribosyltransferase (hprt): 5′-TCCTCCTCAGACCGCTTTT-3′, antisense: 5′-CCTGGTTCATCATCGCTAATC-3′. All qRT-PCR reactions were prepared in 7 μL with final concentrations of 1× Platinum®vSYBR® Green qPCR SuperMix-UDG (Invitrogen), 200 nM primers, using the following cycling conditions: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The starting quantity of the target gene was normalized to the starting quantity of the housekeeping gene hprt.

Statistics

EAE development between experimental groups was analyzed using the “Testing between Curves” permutation test developed by the bioinformatics division of the Walter and Eliza Hall Institute (http://bioinf.wehi.edu.au). Data were presented as means ± SEM. Two-way ANOVA was used to analyze proliferation assay and the Mann–Whitney U-test was used for ELISA. p-values ≤ 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

The timing of bone marrow transfer following treosulfan treatment influences grafting efficiency

Preliminary studies based on the protocol of van Pel (22,23) and involving BM transfer within 24 h of treosulfan treatment resulted in high mortality rates (data not shown). To determine if the timing of BMT could improve mouse survival, we assessed mice receiving BM at 48, 72 and 96 h following the last injection of treosulfan. Control groups included nonmanipulated mice, conditioning with TBI (2 × 550 cGy) and mice that received no BM following treosulfan. Mice preconditioned with TBI displayed some weight loss that recovered with time (Figure 1B). The myeloablative effect of high dose (6000 mg/kg) treosulfan treatment was evident in mice that did not receive BMT, displaying steady weight loss without recovery (Figure 1C). Mice receiving BMT at 48 h also displayed severe weight loss with 2/3 mice requiring euthanasia (Figure 1D). However, mice that received BMT at 72 or 96 h displayed some initial weight loss, which recovered to normal and was indicative of successful grafting (Figure 1E and F).

image

Figure 1. Delay in bone marrow transfer following treosulfan administration is required for successful bone marrow grafting. C57BL/6 mice were treated with a total dose of 6000 mg/kg treosulfan over three injections and transplanted with 7 ×104 cells transduced with retrovirus encoding MOG and GFP. Body weight was monitored daily and represented as a percentage of starting weight. (A) Naive mice, (B) total body irradiated mice, (C) mice not receiving bone marrow, (D) 48 h delay from final treosulfan injection to BMT, (E) 72 h delay from final treosulfan injection to BMT and (F) 96 h delay from final treosulfan injection to BMT. BMT = bone marrow transplantation, TBI = total body irradiation, Treo = treosulfan injections.

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Transduction of BM with a retrovirus encoding MOG and GFP enabled chimerism (GFP+ cells) to be readily detected and analyzed, and at 10 weeks posttransfer (Figure 2A and B) similar levels of chimerism were found in the 72 and 96 h cohorts. This was less than the chimerism observed in the sole survivor of the 48 h group (Figure 2A and B) and any significance of this is difficult to suggest given this single observation and the fact that variation is often observed between individuals within cohorts. Quantitative RT-PCR analysis confirmed MOG expression in all groups and significantly elevated compared to normal mice (Figure 2C). From these data we concluded that 72 h should elapse between treosulfan and BMT to ensure mouse survival, chimerism and MOG expression.

image

Figure 2. Chimerism is generated in lymphoid cells with treosulfan conditioning and associated with elevated MOG expression. Mice surviving from treatment with 6000 mg/kg treosulfan following BMT (Figure 1) were analyzed for chimerism (GFP expression). At 10 weeks, single cell populations from thymus (A) and spleen (B) were stained for cell surface markers and analyzed for GFP expression. (C) Whole splenocytes were subjected to q-PCR to assess relative MOG mRNA levels in the different treosulfan treatment groups compared to normal mice. Data are presented as mean ± SEM. *p < 0.05.

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Lower doses of treosulfan can promote chimerism and immune tolerance

Having established the need for a 72 h time buffer between treosulfan and BMT, we next determined the effect of lower doses of treosulfan on chimerism and susceptibility to EAE. Blood cell analysis 5 weeks post-BMT revealed high levels of chimerism in both MOG and ProII groups generated with the myeloablative dose of 6000 mg/kg treosulfan (Figure 3A). In contrast, the chimerism generated at 4500 mg/kg had a large spread that ranged from 2% to 67% whereas at 3000 mg/kg they were all low but still detectable (Figure 3A). At 1500 mg/kg treosulfan, GFP was no longer detected. Survival of mice treated with these doses of treosulfan without BM rescue confirmed 6000 mg/kg as myeloablative and 4500, 3000 and 1500 mg/kg as nonmyeloablative (data not shown) (28).

image

Figure 3. Chimerism is induced with nonmyeloablative doses of treosulfan and can protect from EAE development. C57BL/6 mice were treated with various doses of treosulfan (1500, 3000, 4500 and 6000 mg/kg) and transplanted with 2–4 × 105 bulk transduced BM cells 72 h after the final treosulfan injection. Mice were bled at 5 weeks and analyzed by flow cytometry for GFP expression. Normal mice and mice receiving MOG transduced BM alone were included as controls. MOG: myelin oligodendrocyte glycoprotein. ProII: proinsulin II. (A) Level of chimerism determined by peripheral blood GFP expression. (B) Eight weeks following bone marrow transfer, mice immunized with MOG35–55 peptide and monitored for EAE development. Clinical scores for each mouse were recorded and mean clinical score ± SEM for each group is shown. *p < 0.05. (C) Kaplan–Meier curve illustrating the percentage of mice in each group that did not develop signs of EAE.

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Eight weeks following BMT, mice were challenged with MOG35–55 peptide to induce EAE. Mice treated with 6000 and 4500 mg/kg treosulfan were resistant to EAE induction and remained EAE free over the 55-day observation period. In contrast, control C57BL/6 mice or preconditioned mice transplanted with ProII BM or mice receiving MOG BM alone developed EAE (Figure 3B and C). The majority of mice that received lower doses of 3000 and 1500 mg/kg treosulfan also developed EAE (Figure 3B and C).

Fresh cohorts of mice generated with the nonmyeloablative dose of 4500 mg/kg confirmed MOG transduced BM conferred EAE resistant, in contrast to control groups (Figure 4A). Peripheral blood analysis at 5 weeks posttransplant revealed chimerism in MOG and ProII chimeric mice was similar and ranged from 0.2 to 12% (Figure 4B). Splenocytes from MOG chimeric mice failed to respond to MOG peptide in vitro (Figure 4C), whereas control mice with EAE or ProII chimeric mice responded to MOG35–55 in a dose-dependent manner (Figure 4C). Lymphocytes from all groups responded to nonspecific concanavalin A stimulation (not shown). Chimeric mice encoding MOG also demonstrated a lack of autoantibodies to MOG35–55 compared to EAE control mice and ProII mice, which developed autoantibody responses to various degrees (Figure 4D).

image

Figure 4. Low levels of chimerism are sufficient to prevent EAE induction and T cell responses. (A) C57BL/6 mice were preconditioned with 4500 mg/kg treosulfan and transferred with BM cells transduced with retrovirus encoding MOG or ProII. Following engraftment, mice were immunized and monitored for development of EAE. Data are shown as mean ± SEM. (B) At 5 weeks, whole blood was analyzed by flow cytometry for GFP expression to monitor engraftment and chimerism. Data are shown as chimerism for each individual mouse and mean ± standard deviation (C) At completion of the experiment, splenocytes were isolated and stimulated in vitro with MOG35–55 peptide. Proliferation was assessed by 3H-thymidine uptake and the degree of proliferation presented as stimulation index. Data shown as mean ± SEM. *MOG versus C57BL/6 EAE/ProII groups, p < 0.05. (D) At the completion of the experiment, sera from mice was collected and assayed for MOG35–55 autoantibodies by ELISA. Sera was analyzed at 1:100 dilution and the degree of antibody reactivity was measured as optical density. Data shown as mean ± SEM. *p < 0.05.

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Nonmyeloablative treosulfan conditioning promotes tolerance and long-term remission in an established model of EAE

To mimic a clinical scenario, we tested whether this strategy could be used to reverse established EAE in a similar manner to our previous study (15). We have shown in mice with EAE that treatment with methylprednisolone promotes remission that relapses upon removal (24). Mice with established EAE (clinical score of 2) were generated and treated with methylprednisolone to promote remission (24). Mice in remission (score of 0) for at least 3 days were preconditioned over 3 days with 4500 mg/kg treosulfan, at which point prednisolone treatment was ceased and BMT performed. Mice also received a single dose of anti-thy1.1 antibody to deplete residual MOG reactive T cells. Groups included normal BMT, MOG or ProII transduced BM and mice conditioned by TBI and receiving MOG BM as a positive control (15) (Figure 5A). Mice were bled at 5 weeks to confirm chimerism (Figure 5B) and observation over an 8-week period revealed none of the mice from any of the groups relapsed with EAE (Figure 5A). At face value, this suggested the protocol of BMT alone was sufficient to promote tolerance. To assess if this was true, mice were rechallenged with MOG35–55 peptide and monitored for relapse. Both groups of mice conditioned with treosulfan or TBI and transplanted with BM encoding MOG maintained resistance following rechallenge (Figure 5A). However, control groups receiving normal BM or BM encoding ProII developed EAE upon rechallenge (Figure 5A). Mice treated solely with 4500 mg/kg treosulfan or anti-thy1.1 antibody also developed EAE, indicating these treatments did not influence disease susceptibility (Figure 5A insert).

image

Figure 5. Long-term remission is maintained following transfer of transduced bone marrow with nonmyeloablative conditioning. (A) C57BL/6 mice were immunized with MOG35–55 peptide and monitored for development of EAE. Mice displaying a clinical score of 2 (limp tail and hind limb weakness) were treated with methylprednisolone to promote remission and stopped prior to BMT. Mice were preconditioned with 4500 mg/kg treosulfan and transferred with BM cells transduced with retrovirus encoding MOG or ProII or nonmanipulated BM. All mice also received a single dose of depleting anti-thy1.1 antibody at time of BM transfer. Mice were monitored for EAE development and at day 91, mice were re-immunized with MOG35–55 peptide and monitored for EAE induction. Insert demonstrates that mice treated with anti-thy1.1 or treosulfan alone are not resistant to EAE induction. (B) At 5 weeks, mice were bled and GFP expression was assessed as measure of chimerism. Chimerism for each individual mouse is shown as well as mean ± SEM.

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Histology of spinal cords showed that mice receiving MOG transduced BM were free of inflammatory infiltrates (Figure 6A and B) and similar to normal mice (Figure 6H). In contrast, mice that received normal BM or BM encoding ProII (Figure 6A,C,D) displayed inflammatory infiltrates similar to that observed in control mice with EAE (Figure 6A,E). Mice that received anti-thy1.1 or treosulfan alone were not protected and also displayed infiltrates (Figure 6F,G).

image

Figure 6. Mice with EAE and treated with bone marrow cells encoding MOG are protected from disease relapse. Mice with EAE were treated with methylprednisolone, preconditioned with a nonmyeloablative dose of treosulfan and transferred with BM encoding MOG, ProII or nonmanipulated BM as outlined in legend to Figure 5. (A) At completion of the experiment, spinal cords were collected, fixed in formalin and analyzed by hematoxylin and eosin staining for cellular infiltrate. Sections were scored for infiltrate and data are presented as mean histological score ± SEM. Representative images (B–H) of spinal cord are shown for mice receiving BM encoding MOG (B), normal BM (C), irrelevant autoantigen ProII (D), mice treated with anit-thy1.1 alone (F), mice treated with treosulfan alone (G) and spinal cord from a normal mouse (H). Images were captured at × 20 magnification.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Autologous HSCT as a treatment for autoimmune disease involves a process aimed at ablating pathogenic clones and “resetting” of the immune system. While benefits are reported, relapse rates are significant (5,7,29), suggesting that tolerance with HSCT alone has not been established. Our studies support this and mice transferred with syngeneic BM are not resistant to EAE induction (15), confirming that HSCT alone does not promote tolerance. The transfer of BM cells transduced with retrovirus encoding autoantigen can promote antigen specific tolerance (14,15), and involves a process similar to that currently used to treat human immunodeficiencies (30,31); namely ex vivo retroviral manipulation and transfer of autologous BM. A key consideration in utilizing HSCT for treating autoimmunity is the preconditioning regime and the ability to use lower toxic and nonmyeloablative regimes to minimize treatment related mortality (5). Experimental models to date have predominantly used lethal TBI to establish that the transfer of BM encoding autoantigen can be used to impart tolerance (14,15,17). Fewer studies have examined nonlethal doses (14,19), and no studies have utilized chemotherapeutic agents in scenarios that might mimic a clinical situation. To extend our current knowledge in this area, we adopted our EAE model for treatment with treosulfan; a water-soluble analogue of busulfan that is used as a preconditioning agent in animals (23,28) and pediatric HSCT (32).

The protocol of van Pel (22) we initially utilized resulted in high mortality in our mice and suggested graft failure. High mortality was not an issue for van Pel and subsequent experiments demonstrated that transferring BM within 48 h of the last treosulfan injection was responsible and likely due to residual treosulfan within the mice. Studies in paediatric patients conditioned with treosulfan prior to allogeneic HSCT showed that only 30% of the drug is eliminated within the first 12 h (33) suggesting that BM transfer too soon after treosulfan might indeed expose BM cells to its toxicity. Another factor which may have contributed to the sensitivity observed in our study is the fact that we transferred 200-fold less cells than the 1.5 × 107 cells transferred by van Pel (22). This was a technical and practical necessity given the in vitro culturing and manipulation we were performing on collected BM. By delaying the transfer of BM to 72 h, we could eliminate mortality and maintain the capacity to generate chimeric mice with fewer cells. This information may be of use to others wishing to utilize nonmyeloablative conditioning regimes and BMT in their studies.

In testing a range of treosulfan doses, we found that chimerism could be achieved at the nonmyeloablative dose of 4500 mg/kg. While there was a large spread in chimerism, regardless of the level none of the mice developed EAE. The average level of chimerism at this dose was approximately 8% and much lower than the 60% reported by van Pel, (22,23). This difference most likely reflects differences between the systems and in particular the larger dose of cells transferred by van Pel. In addition, our protocol probably underestimates the true degree of BM grafting since our criteria for chimerism is GFP+ cells and thus stem cells that have not been transduced may still contribute to grafting but will not be detected. To address this, we would need to utilize congenic markers such as Ly5.1/5.2 to enable differentiation of donor and recipient cells. However, and more importantly, our studies indicate that chimerism in the order of a few percent is sufficient to promote EAE resistance, with T cell tolerance and absence of autoantibody production. These findings are similar to our earlier studies in mice generated with lethal irradiation and higher levels of chimerism achieved (15). The observation in the 3000 mg/kg group that developed extremely low chimerism and 2/5 mice remained resistant to EAE is tantalizing; although an immediate correlation between chimerism and EAE was not obvious and a larger study will need to determine if microchimerism may be involved. The finding that only low levels of chimerism are required to induce tolerance is not new in itself and Steptoe and colleagues, transferring BM from transgenic mice encoding proinsulinII, reported that 5% chimerism could prevent development of type 1 diabetes (34). Similarly, 1% donor chimerism is sufficient for transplant tolerance (35,36) and demonstrated in an elegant study utilizing BM encoding the MHC class I molecule Kb and skin graft acceptance (37). Collectively, all these findings reinforce the notion that the bone marrow compartment has a powerful influence on the induction of immunological tolerance that can be harnessed to address a range of disease conditions.

We extended the study to demonstrate the feasibility of using nonmyeloablative conditioning in treating mice with established EAE and reinforced the notion that only a few percent chimerism is required. This has important implications for human translation where the degree of toxicity associated with the procedure will be a major factor. In essence we have demonstrated that we could adopt similar protocols used in human HSCT treatment for autoimmunity to promote immune tolerance. While we emphasize the need of autoantigen expression for tolerance, the protocol involved a number of steps including corticosteroid treatment, chemotherapeutic preconditioning and antilymphocyte antibodies that would contribute to depleting existing pathogenic clones. This is a similar process to humans undergoing autologous HSCT to treat multiple sclerosis (3,38), and in itself may provide some measure of benefit. Indeed, we also observe in our studies that mice with established EAE remained in remission following normal BM transfer. However, we stress that BMT alone does not induce tolerance and mice remained susceptible, which was easily demonstrated by rechallenge with MOG.

The key experiment in this study utilized established EAE in showcasing the feasibility of this strategy in a clinically relevant manner. There are few studies that have examined established EAE with our own studies using lethal irradiation as a forerunner (15). Xu and colleagues (14) using low dose irradiation as a means of nonmyeloablative conditioning reported in an EAE study some protection if transduced BM was given within 12 days of immunization but this had no effect if given on day 22; when EAE would be well established. However this study did not include any corticosteroid or antilymphocyte steps and underpins the importance that these procedures may have on the overall outcome. The mechanism of tolerance in this study was not defined but we have described in earlier studies that thymic deletion is active (15). However, given that antigen is expressed in a variety of bone marrow derived cells, it is possible that peripheral mechanisms may also be active and we will need to define these in further studies. In summary, these studies have shown that utilizing nonmyeloablative conditioning together with BMT creates low level chimerism that can impart autoantigen specific immunological tolerance that incorporated into a clinically protocol can be used to cure an established autoimmune disease.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

These studies were supported by funding from the Australian National Health and Medical Research Council. ZN is recipient of an Australian Postgraduate Award. JYC is recipient of Multiple Sclerosis Research Australia postgraduate award.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

The authors have no conflicts of interest to disclose as required by the American Journal of Transplantation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
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    Rose NR, Mackay IR. The Autoimmune Diseases, 4th Ed. San Diego , California : Elsevier Academic Press, 2006.
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