Short-course rapamycin treatment enables engraftment of immunogenic gene-engineered bone marrow under low-dose irradiation to permit long-term immunological tolerance
© The Author(s). 2017
Received: 21 December 2016
Accepted: 11 February 2017
Published: 9 March 2017
Application of genetically modified hematopoietic stem cells is increasingly mooted as a clinically relevant approach to protein replacement therapy, immune tolerance induction or conditions where both outcomes may be helpful. Hematopoietic stem and progenitor cell (HSPC)-mediated gene therapy often requires highly toxic pretransfer recipient conditioning to provide a ‘niche’ so that transferred HSPCs can engraft effectively and to prevent immune rejection of neoantigen-expressing engineered HSPCs. For widespread clinical application, reducing conditioning toxicity is an important requirement, but reduced conditioning can render neoantigen-expressing bone marrow (BM) and HSC susceptible to immune rejection if immunity is retained.
BM or HSPC-expressing OVA ubiquitously (actin.OVA) or targeted to MHC II+ cells was transferred using low-dose (300 cGy) total body irradiation. Recipients were administered rapamycin, cyclosporine or vehicle for 3 weeks commencing at BM transfer. Engraftment was determined using CD45 congenic donors and recipients. Induction of T-cell tolerance was tested by immunising recipients and analysing in-vivo cytotoxic T-lymphocyte (CTL) activity. The effect of rapamycin on transient effector function during tolerance induction was tested using an established model of tolerance induction where antigen is targeted to dendritic cells.
Immune rejection of neoantigen-expressing BM and HSPCs after low-dose irradiation was prevented by a short course of rapamycin, but not cyclosporine, treatment. Whereas transient T-cell tolerance developed in recipients of OVA-expressing BM administered vehicle, only when engraftment of neoantigen-expressing BM was facilitated with rapamycin treatment did stable, long-lasting T-cell tolerance develop. Rapamycin inhibited transient effector function development during tolerance induction and inhibited development of CTL activity in recipients of OVA-expressing BM.
Rapamycin acts to suppress acquisition of transient T-cell effector function during peripheral tolerance induction elicited by HSPC-encoded antigen. By facilitating engraftment, short-course rapamycin permits development of long-term stable T-cell tolerance.
KeywordsBone marrow transplant Gene therapy Immune tolerance
Gene therapy approaches employing genetically modified hematopoietic stem and progenitor cells (HSPCs) show great promise for expression of therapeutic proteins within the hematopoietic system. Notable clinical successes have been achieved with therapy of severe combined immunodeficiency (scid)-X1 , leukodystrophies [2, 3] and Wiskott–Aldrich syndrome . Potential applications tested in preclinical models are more diverse, encompassing a range of blood disorders including hemophilia and sickle-cell disease  and immunological disorders .
Enforced expression of antigen either ubiquitously or targeted to antigen-presenting cells (APC) of the immune system is a robust approach to inducing immune tolerance which prevents priming of T-cell and B-cell responses to expressed proteins [7, 8]. This has the power to prevent development of autoimmune disease in elicited and spontaneous models leading to proposals for therapeutic application [9, 10]. Indeed, under certain conditions established memory T-cell responses can be turned off , suggesting that enforced antigen expression may provide unique opportunities to control otherwise difficult-to-treat memory T-cell responses. For therapeutic application, an efficient means to achieve de-novo enforced antigen expression is HSPC-based gene therapy .
A critical requirement for clinical application of HSPC-based gene therapies, particularly if intended for therapy of nonlife-threatening diseases, is to minimise the toxicity of procedures associated with HSPC transfer. Currently for HSPC-mediated gene therapies, highly toxic pretransplant conditioning that both myeloablates and immunoablates patients prior to HSPC transfer is typically used [3, 4]. This facilitates high levels of engraftment of transferred HSPCs along with substantial replacement of recipient hematopoietic cells with those derived from the transferred, engineered HSPCs respectively. In some disorders, such as those where substantial replacement of long-lived hematopoietically derived cells might be required, immunoablation may be advantageous [4, 13], but may not be required in, for example, scid, where progeny of engineered HSPCs have a competitive advantage and could more easily repopulate recipients . In other disorders where large-scale replacement of immune cells is not required, immunoablation is disadvantageous because it would be preferable to preserve existing protective immunity. However, depending on the approach used for directing expression of therapeutic proteins, preserving recipient immunity renders transferred gene-engineered HSPCs susceptible to immune attack  and failure of engraftment if they express neoantigens as a result of engineering. This is of relevance for both protein replacement therapies and approaches for instatement of immune tolerance, for example.
Previously we showed that one approach to limiting immune attack and preserving the integrity of transferred gene-engineered bone marrow (BM) and HSPCs was to restrict neoantigen expression to differentiated leukocytes, away from engrafting HSPCs . Ubiquitous or ‘off-target’ expression of neoantigens in BM or HSC leads to their destruction in recipients with intact immunity [15, 16]. An alternative approach may be to limit the development of immune responses in HSPC recipients.
Here we continue to explore avenues to overcome immune resistance to engraftment of neoantigen-expressing gene-engineered HSPCs. Rapamycin is an immunosuppressant that functions by inhibiting mammalian target of rapamycin (mTOR) to block entry of T cells into the cell cycle which, unlike the calcineurin inhibitors cyclosporine and tacrolimus, does not inhibit TCR-induced Ca2+ signaling [17, 18], which is important for tolerance induction in some conditions [19, 20]. Both clinical and preclinical studies indicate that rapamycin is ‘tolerance-permissive’ in organ allograft and other settings, whereas cyclosporine and tacrolimus may inhibit the development of immune tolerance . We report that a short course of rapamycin treatment is sufficient to prevent immune rejection of neoantigen-expressing BM and HSPCs in recipients with intact immunity. Protection of engrafting cells is mediated by suppression of transient effector differentiation in T cells undergoing peripheral tolerance induction elicited by HSPC-encoded antigen.
OT-I, 11c.OVA, MII.OVA and actin.OVA mice have been described elsewhere [7, 22–24]. Mice were maintained under specific pathogen-free conditions in the TRI Biological Resources Facilities, Brisbane, Australia. Nontransgenic C57BL/6 and B6.SJL-PtprcaPep3b/BoyJArc (B6.SJL) mice were purchased from ARC (Perth, Australia). Unless stated otherwise, in BM transplant experiments recipient mice were B6.SJL (CD45.1+) and donors were MII.OVA (CD45.2+), actin.OVA (CD45.2+) or nontransgenic (non-Tg) C57BL/6JArc (all CD45.2+) mice. To generate CD45.1+/CD45.2+ OT-I mice, B6.SJL mice were crossed with OT-I mice. All animal procedures were performed in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes and approved by the University of Queensland Animal Ethics Committee (projects DI/208/12; UQDI/296/14).
Bone marrow and HPC transplantation
Donor mice were euthanised by CO2 narcosis and femurs and tibias collected into mouse tonicity (MT)-PBS. Bone marrow was flushed with MT-PBS/2.5% FCS, erythrocytes lysed (NH4Cl/TRIS buffer) and BM washed twice (MT-PBS/2.5% FCS). BM was resuspended in MT-PBS and injected i.v. (lateral tail vein) within 3 hours of total body irradiation (TBI; 300 cGy, 137Cs source). Unless stated otherwise, 10 × 106 bulk BM was transferred. For high-dose irradiation experiments, the irradiation was delivered as two equal doses (550 cGy) 3 hours apart and mice were administered neomycin (1 mg/ml) in drinking water for 3 weeks. HSPCs were prepared by high-speed FACS sorting of lin–ve/c-kit+ve cells to typically >95% purity from bulk BM. HSPC-depleted BM was lin+ve/c-kit–ve cells prepared from BM by high-speed cell sorting.
Rapamycin (Rapamune, Wyeth Australia) was diluted in PBS and administered (0.6 mg/kg) by i.p. injection. Cyclosporine (Sandimmune, Novartis Pharmaceuticals Australia) was diluted in PBS and administered (25 mg/kg) daily by i.p. injection. Immunosuppressant administration commenced on the day of BM/HSPC transfer and continued daily for the following 21 days unless the experiment finished sooner. To determine the blood concentration of rapamycin, whole blood was collected in 0.5 M EDTA immediately prior to rapamycin administration on the days indicated and stored at −30 °C. LC-MS/MS analysis was performed using an Alliance HT LC system interfaced to a Quattro-Premier mass spectrometer (Waters Corporation, Milford, MA, USA).
In-vitro and in-vivo assays
OVA/QuilA immunisation was as described previously . Intracellular cytokine staining and in-vivo CTL assays were performed as described previously . CFSE labeling was performed as described elsewhere  and proliferation indices calculated as described previously .
Sample preparation for flow cytometry of BM, spleen and pooled lymph node (axillary, brachial, inguinal and mesenteric) was as described previously . mAb were purchased from Biolegend, BD Biosciences and BioXcell (Lebanon, NH, USA) or were grown, purified and conjugated in-house. Analysis of peripheral blood for engraftment determination was performed using a bead-based counting assay as described previously . Cytometric data were acquired using BD Canto or BD LSRII cytometers and analysed using Diva (BD) or Flow-Jo (Tree-Star) software.
Student’s t test (two-tailed) was used for comparison of means and one-way ANOVA with Newman–Keuls or Tukey’s post test for multiple comparisons (GraphPad Prism 5 or Prism 6). p < 0.05 was considered significant.
Increasing the dose of cells injected partially overcomes immune resistance to gene-modified BM
Short-course rapamycin treatment permits engraftment of neoantigen-expressing BM under immune-retaining conditioning
Rapamycin promotes engraftment by limiting immune rejection
As shown in Fig. 1 controlling immune pressure by administration of rapamycin allows MII.OVA BM to engraft stably, but at a consistently reduced level compared with non-Tg or actin.OVA BM (Fig. 1c–j). This likely reflects reduced engraftment capacity of MII.OVA HSC  but could also potentially reflect transgene expression-induced endoplasmic reticulum (ER) stress  that might be relieved by rapamycin. Therefore, we tested the effect of rapamycin on the fitness of MII.OVA and actin.OVA BM in a competitive repopulation assay. Because recipient immunity is ablated by the lethal irradiation used, nonimmune effects of rapamycin are tested. When equal numbers of actin.OVA and non-Tg control or MII.OVA and non-Tg control BM were mixed and transferred to high-dose (1100 cGy) irradiated mice, MII.OVA BM showed a deficit compared with actin.OVA BM in leukocyte accumulation and engraftment in the HSPC compartment in PBS-treated controls (Fig. 2c, d) as expected (Fig. 1 ), consistent with reduced hematopoietic capacity. Notably, administration of rapamycin did not alter the relative pattern of donor-type leukocyte development in or between recipient groups (Fig. 2c, d), indicating that rapamycin did not provide a competitive advantage specific for MII.OVA engraftment and hematopoiesis in the absence of immune pressure.
Antigen-expressing BM transfer paradoxically induces both BM rejection and transient T-cell unresponsiveness to BM-expressed antigen in the absence of rapamycin
Stable long-term tolerance to BM-expressed antigen requires rapamycin-facilitated engraftment
Before investigating the mechanisms that might underlie failure of CTL induction 4 weeks after BM transfer, we wished to determine firstly whether unresponsiveness was transient, indicating a peri-BMT effect, or whether unresponsiveness to OVA was long-lasting irrespective of rapamycin administration. To achieve this we performed BM transfers, but waited 25 weeks before mice were sham-immunised or immunised with OVA/QuilA and then OVA-specific in-vivo CTL activity was tested 1 week later. In this setting, in recipients of non-Tg BM regardless of rapamycin treatment there was strong CTL activity, equivalent to that in no BMT controls, induced by immunisation. In MII.OVA BM recipients, immunisation elicited CTL activity only in PBS-treated but not rapamycin-treated recipients (Fig. 3b). A similar trend was observed in the small number of actin.OVA BM recipients analysed (Fig. 3b); however, this was not significant in the PBS-treated group due to the low number of mice tested. In contrast, in rapamycin-treated recipients of MII.OVA and actin.OVA BM, CTL induction by immunisation was almost completely damped (Fig. 3b). This demonstrates that although there was modulation of OVA responsiveness soon after BMT in recipients of OVA-encoding BM regardless of rapamycin treatment, stable, long-lasting T-cell tolerance only occurred when OVA-expressing BMT was combined with rapamycin to facilitate stable engraftment of OVA-expressing BM.
Rapamycin treatment delays T-cell recovery after BMT
To investigate why OVA responsiveness may have been modulated soon after BMT in rapamycin-treated non-Tg BM recipients and perhaps in OVA-encoding BM recipients, we first determined whether residual rapamycin may play a role. The rapamycin concentration in whole blood was within the clinical therapeutic range (6–15 μg/l) in most mice throughout the treatment period but had diminished to undetectable levels 7 days after cessation of treatment (Fig. 3c) when mice in some experiments mice were immunised. Examination of the T-cell repopulation kinetics after irradiation showed that rapamycin administration delayed recovery of CD8+ and CD4+ T-cell populations from the partial lymphopenia induced by low-dose irradiation (Fig. 3d, e). This was most prominent during or soon after cessation of rapamycin treatment (Fig. 3d, e). By 5 weeks after rapamycin cessation, T-cell recovery was approximately equal in all groups. This suggests that rapamycin-treated recipients of non-Tg BM exhibit impaired antigen-responsiveness due to reduced T-cell repopulation. However, this reflects total CD8+ or CD4+ T-cell number and does not necessarily indicate relative repopulation with OVA-specific T cells or OVA responsiveness specifically, nor explain why PBS-treated recipients of OVA-encoding/expressing BM exhibit CTL unresponsiveness 4–5 weeks after BMT. When the presence of CD4+CD25+FoxP3+ regulatory T cells (Treg) was analysed, we found no evidence that Treg were preferentially expanded in any group, suggesting that Treg were not responsible for the unresponsiveness observed.
Development of stable long-term tolerance engraft is associated with the extended presence of cognate antigen
OVA arises from transferred HSPCs in MII.OVA BM recipients
Paradoxically, in the absence of rapamycin, transfer of OVA-expressing BM induces not only transient tolerance but also promotes the ultimate rejection of engrafting HSPCs. It is possible that this unresponsiveness is due to transient expression of OVA in PBS-treated recipients of OVA-expressing BM because immunologically active OVA is not present 26 weeks after BMT (Fig. 4a, d). Because cells within the transferred whole BM prepared from MII.OVA donors express OVA  and could potentially induce tolerance or immunity , we next determined whether the immunologically relevant OVA present in PBS-treated MII.OVA BM recipients that might have contributed to induction of rejection and the development of unresponsiveness to OVA was derived from the BM graft, HSPCs within the graft or from the non-HSPC component of the graft. Whole BM, HSPCs or HSPC-depleted BM from MII.OVA and BM from non-Tg mice was transferred to low-dose irradiated recipients with or without rapamycin treatment and 4 weeks after BMT CFSE-labeled OT-I T cells were transferred to detect the presence of immunologically relevant OVA. OT-I proliferated only in recipients of MII.OVA whole BM or HSPCs and not HSPC-depleted BM (Fig. 4d). This suggests the source of OVA in MII.OVA BM recipients was HSPCs, most likely through engraftment and/or development of progeny (Fig. 1). Based on the reduced proliferation of OT-I T cells at week 5 (Fig. 4b) relative to week 4 (Fig. 4e), residual OVA is cleared quickly after engraftment failure in PBS-treated recipients.
Rapamycin inhibits development of transient effector function during tolerance induction
To identify whether the action of rapamycin was to inhibit differentiation of effector function in the ‘intrinsically tolerogenic’ MII.OVA BM transfer setting, non-Tg or MII.OVA BM was transferred to low-dose (300 cGy) irradiated recipients with or without rapamycin administration, and spontaneous CTL activity that had developed 17 days later, when BM rejection has commenced (Fig. 1, Additional file 1) and engraftment is failing (Fig. 4c), was tested. No CTL activity was detected in recipients of non-Tg BM as expected (Fig. 5g). In contrast, in PBS-treated MII.OVA BM recipients, substantial killing of OVA257–264-pulsed targets was observed, which was not present in rapamycin-treated MII.OVA BM recipients (Fig. 5g).
Rapamycin therefore acts to prevent the emergence of the transient effector function elicited in OVA-specific T cells as a component of tolerance induction. Ironically, engraftment appears to proceed initially giving rise to donor-derived antigen expressing DC (Additional file 1) and while these are potentially tolerogenic, the transient effector function elicited in the early phase of tolerance induction leads to rejection of antigen-expressing HSC. In the absence of transient immune suppression to control this, engraftment fails and induction of long-term tolerance that requires ongoing antigen expression by the progeny of successfully engrafted OVA-encoding HSPCs stalls before fully developing.
Increasing the clinical applicability of HSPC-based gene therapy is an important goal that will maximise the usefulness of this potentially powerful therapeutic. Defining approaches that reduce the toxicity of HSPC transfer-associated procedures is a key requirement. Enabling high levels of engineered HSPC engraftment and subsequent leukocyte development through either increasing the competitive advantage of transferred engineered HSPCs or opening ‘engraftment niches’ in the recipient whilst reducing treatment toxicity is one important focus. However, here we have focused on overcoming the challenges of immune resistance that is a consequence of attempts to achieve the desired outcome of preserving recipient immune function during HSPC-mediated gene transfer. Here we show that HSPC-based approaches capable of inducing immune tolerance which could, for instance, alleviate autoimmune diseases or allergies can be hindered by the development of transient effector function in the very T cells that are targeted for inactivation by the procedure. Development of transient effector function is a normal component of the early phase of tolerance induction in T cells [23, 30, 31] but, using rapamycin, we show this can be readily controlled by a short course of appropriate immunosuppressant administration. Limiting effector differentiation during the critical peritransfer period facilitates engraftment and leads to establishment of long-term tolerance that does not require additional immunosuppression for maintenance.
A notable observation was that rapamycin was highly effective at promoting engraftment of BM expressing a neoantigen under the immune-preserving conditions used, but that cyclosporine was much less effective. This is supported by similar results in an allogeneic BM transplant setting . Competitive repopulation assays ruled out that rapamycin provided a nonimmunological engraftment-enhancing effect to transgene-encoding HSPCs. While rapamycin did not appear to act on HSPCs, agents that protect the HSPC niche from radiation-induced damage or foster hematopoiesis or myelopoiesis/erythropoiesis, such as lysophophatidic acid [33, 34], might promote post-HSPC transfer engraftment. Rapamycin or its analog everolimus has been reported as ‘tolerance-permissive’ in organ allograft  and other settings [35, 36], and it is possible that this underlies these observations. Effectiveness here, however, appeared to be associated with the capacity of rapamycin to inhibit both expansion and transient effector function elicited by tolerogenic antigen presentation. Cyclosporine, by contrast, poorly controlled expansion of T cells undergoing tolerance induction. In a small number of cyclosporine-treated animals tested, responsiveness to OVA was inversely correlated with the level of MII.OVA BM engraftment present. This is in line with previous conclusions that, under conditions where potentially tolerogenic BM is transferred, tolerance is related to successful engraftment  rather than perhaps the immunosuppressant used. It might be that the effectiveness of rapamycin as an anti-proliferative agent for T cells  is the critical factor, particularly here where tolerogenic rather than immunogenic antigen presentation is present. The anti-proliferative effects of rapamycin impaired T-cell recovery after irradiation and BM transfer, and this could potentially also contribute. Interestingly, the extent of the rapamycin-induced delay in T-cell reconstitution differed somewhat between CD8+ and CD4+ T cells. Why CD4+ T cells appear to be more affected remains unclear. However, a possible explanation is that the homeostatic proliferation which contributes to T-cell recovery after low-dose irradiation  is modulated by the differential sensitivity of distinct homeostatic cytokines such as IL-7 and IL-15 to rapamycin-mediated inhibition of mTOR between different T-cell subsets [39–41].
Moderate doses of irradiation can lead to BM transfer-associated regulatory T cell (Treg) expansion  which could potentially be enhanced by rapamycin. While not shown, we found no evidence that Treg expansion contributed to rapamycin-mediated effects. However, in other studies the irradiation dose required for expansion of antigen-specific CD4+CD25+FoxP3+ Treg was higher (>450 cGy)  or myeloablative doses of irradiation were used for CD8+Foxp3+ Treg induction/expansion , and the latter study used an allogeneic transplant setting and alloantigen was required for Treg generation/expansion. In other studies exploring transfer of antigen-encoding BM, no evidence of Treg induction has been reported  unless CD4+ TCR transgenic T cells are included [44, 45]. Administration of rapamycin has also been shown to induce or expand Treg in vivo, but in many cases this has been in the presence of coadministered antigen and/or a source of exogenous IL-2 [35, 36] and strong inflammatory signals may promote this effect . The rapamycin treatment period of 3 weeks chosen here was based on our previous studies showing that induction of peripheral CD8+ T-cell tolerance is complete within 2–3 weeks of antigen encounter  and, although not tested here, it is possible a shorter course is also effective.
In the absence of rapamycin, engraftment and leukocyte development is transient, proceeds for approximately 2 weeks, but ultimately fails (Fig. 1) due to immune rejection. Paradoxically, despite immune rejection of OVA-encoding BM, OVA-specific CD8+ T cells are either deleted or rendered antigen-unresponsive as recipients fail to develop CTL activity in response to immunisation for some time after BM rejection. We conclude this is mediated by a transient presence of HSPC-derived OVA manifesting in the absence of rapamycin. However, once OVA is no longer present, immune responsiveness recovers likely through thymic export of OVA-specific T cells which is prevented by central tolerance in the presence of stable OVA-encoding BM engraftment. Whether HSPCs directly, or their progeny or host APC, are responsible is yet to be defined. It is also possible in this setting that the CTL activity elicited against transferred HSPCs is integral to tolerance induction by inducing apoptosis-mediated release of tolerogenic antigen as reported for CTL attack of pancreatic islet β cells .
The transient effector state that occurs in T cells early during tolerance induction [23, 30, 31] likely reflects a partially differentiated state that occurs during peripheral tolerance induction while T cells are integrating environmental signals and the final cell is being determined. The presence of transient T-cell effector function during ‘tolerisation’ is likely of little consequence under normal steady-state conditions because only a small number of potentially pathogenic autoreactive T cells would be undergoing tolerance induction at any one time and the number of target cells would be numerically much larger. Although immune preserving, the conditioning used in the BM transfer setting tested here results in partial lymphopenia which has the potential to promote the deleterious effects of the transient effector function elicited , and this may be particularly evident when target cells, in this case engrafting HSPCs, are present in low numbers. Under these circumstances, controlling transient effector function appears critical and rapamycin may be particularly effective through the combined effects on proliferation and effector differentiation discussed.
Our previous studies and those of others indicate that long-lasting expression of BM-encoded antigen is crucial to maintain tolerance [15, 49, 50]. Our data are consistent with a conclusion that many cellular sources of antigen are tolerogenic, but a critical window exists where the cellular antigen sources require protection from transient effector T-cell attack to establish tolerance. Supporting this there is emerging evidence in humans that a persistent source of antigen maintains BM-induced tolerance, although the source of antigen may not need to be BM-derived cells . Lessons learned here that transient immunosuppression, using appropriate tolerance-permissive agents, provides a window of opportunity for tolerance induction may be applicable to a range of gene-therapy settings where immunity is preserved and the potential for immune resistance to therapeutic proteins is generated. Potential settings include limiting immune responses to therapeutically expressed proteins, facilitating viral vector-mediated gene transfer where the viral vector may be immunogenic approaches or preventing immune responses to the products of genes edited using, for example, CRISPR/Cas9 technologies.
A short course of rapamycin promotes the engraftment of gene-engineered, antigen-expressing BM by suppressing the acquisition of transient T-cell effector function during peripheral tolerance induction that is elicited by HSPC-encoded antigen. By facilitating engraftment, short-course rapamycin permits development of long-term stable engraftment which maintains T-cell tolerance through a combination of central and peripheral mechanisms. Such short-course treatment with conventional immunosuppression represents a clinically applicable approach to overcoming immune resistance to genetically engineered bone marrow when immune-preserving conditions are employed.
Bone marrow transplant
Carboxyfluorescein succinimidyl ester
Cytotoxic T lymphocyte
Hematopoietic stem and progenitor cell
T cell receptor
The authors thank Robert Ling for provision of technical help. The authors would also like to Professor Leonard Harrison (Walter and Eliza Hall Institute), Professor Francis Carbone (University of Melbourne) and Professor Mark Jenkins (University of Minnesota) for providing mice. The authors declare no conflicts of interest.
This work was supported by NHMRC Project Grant GNT1013066 (RJS). KHB was supported by a UQ Postdoctoral Fellowship. RJS was a recipient of Australian Research Council Future Fellowship (FT110100372). JWW was supported in part by grants from the Australian Research Council (DP150103714) and by a Perpetual Trustees Fellowship.
Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files.
KHB and RJS designed and performed experiments, analysed data and wrote the manuscript. RR, JFB, RG and J-WJ performed experiments. JWW provided reagents. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All animal procedures were approved by the University of Queensland Animal Ethics Committee.
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