The A2A receptor agonist CGS 21680 has beneficial and adverse effects on disease development in a humanised mouse model of graft-versus-host disease
Introduction
Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative method for numerous haematological malignancies and other blood disorders; however, HSCT is limited by the development of graft-versus-host disease (GVHD) [1]. GVHD develops in up to 60% of HSCT recipients [2], due to donor T cells recognising ‘foreign’ host cells [3]. GVHD occurs when damage caused by the conditioning regime or the underlying disease promotes the release of inflammatory cytokines such as tumour necrosis factor (TNF)-α and interleukin (IL)-6. Subsequently, activation of CD4+ T cells by dendritic cells (DCs) results in the further release of TNF-α as well as interferon (IFN)-γ, IL-2 and IL-6 to promote inflammation, and subsequent activation of CD8+ T cells to exacerbate this inflammation. Conversely, T regulatory (Treg) cells and invariant natural killer T (iNKT) cells can reduce pro-inflammatory effects in GVHD to limit disease development or progression [4].
Adenosine receptors (A1, A2A, A2B and A3) are cell-surface G-protein coupled receptors activated by extracellular adenosine [5]. Extracellular adenosine is often produced as a result of ATP hydrolysis mediated by the sequential action of ecto-nucleoside triphosphate diphosphohydrolase-1 (CD39), and ecto-5′-nucleotidase (CD73) [6,7]. The A2A receptor is expressed on numerous immune cell subsets including DCs and T cells [8]. Notably, CD39/CD73-mediated production of adenosine and subsequent activation of A2A is an important anti-inflammatory mechanism [9]. In allogeneic mouse models of transplantation, adenosine production by the CD39/CD73 pathway and subsequent activation of adenosine receptors prevents tissue damage and reduces graft rejection [10]. In allogeneic mouse models of GVHD, genetic deficiency or pharmacological blockade of CD73 with αβ-methylene ADP (APCP) [11], which results in reduced extracellular adenosine, worsens disease. Similarly, genetic deficiency [11,12] or pharmacological blockade of A2A with SCH58261 [12] also worsens GVHD severity in these models. Conversely, activation of A2A with ATL-146e can ameliorate GVHD in allogeneic mouse models [13,14]. However, the action of A2A activation in humanised mouse models or HSCT patients remains to be explored.
Allogeneic mouse models are often used to investigate potential therapeutics for GVHD, yet therapies investigated in these models often do not translate to the clinic. This lack of translation is possibly due to species differences. In an attempt to address this, preclinical “humanised” mouse models have been developed [15]. A commonly used humanised mouse model involves the intraperitoneal (i.p.) or intravenous (i.v.) injection of human peripheral blood mononuclear cells (hPBMCs) into immunodeficient non-obese diabetic severe-combined immunodeficiency-IL-2 receptor γnull (NSG) mice. Due to defective T and B cells, and a lack of natural killer (NK) cells, these mice readily engraft hPBMCs [16], and subsequently develop GVHD due to the ability of human T cells to recognise the major histocompatibility complex (MHC) I and II of NSG mice [17]. Previous studies have shown that i.p. or i.v. injection of hPBMCs into these mice results in similar splenic engraftment of human leukocytes [17] and progression of clinical GVHD [18].
Using the A2A agonist CGS 21680 [19], this study aimed to investigate the effect of A2A activation on GVHD development in a humanised mouse model. CGS 21680 did not impact clinical score or survival of mice. However, CGS 21680 reduced leukocyte infiltration into livers, and reduced serum hTNF-α concentrations indicative of reduced GVHD severity. Conversely, CGS 21680 worsened weight loss, reduced Treg cell frequency and increased serum hIL-6 concentrations indicating worsened GVHD. Notably, CGS 21680 also prevented weight gain in NSG mice not engrafted with hPBMCs. This suggests that appetite or metabolism may be negatively impacted by CGS 21680. Therefore, the adverse impact on weight, Treg cells and IL-6 caused by CGS 21680 may confound the potential benefits of A2A activation in reducing GVHD in HSCT recipients.
Section snippets
Humanised mouse model of GVHD
Experiments involving human blood and mice were approved by the respective Human and Animal Ethics Committees of the University of Wollongong (Wollongong, Australia). A humanised mouse model of GVHD was used as described [20]. Briefly, female NSG mice aged 6–8 weeks (Australian BioResources, Moss Vale, Australia) were injected i.p. daily (days −2 to day 11) with saline/0.2% DMSO (Sigma-Aldrich, St Louis, MO, USA) (vehicle) or vehicle containing CGS 21680 (Tocris Bioscience, Bristol, UK)
CGS 21680 does not impact initial hPBMC engraftment in NSG mice
NSG mice injected with hPBMCs and either control vehicle (n = 25) or the A2A agonist CGS 21680 [19] (n = 25) daily (days −2 to 11) were monitored (from day 0) for up to 10 weeks. To determine whether A2A activation affected initial hPBMC engraftment, blood was collected 3 weeks post-hPBMC injection and cells immunophenotyped by flow cytometry. Three mice from each group did not demonstrate human leukocytes (hCD45+mCD45−) in their blood at 3 weeks (results not shown). In the remaining mice,
Discussion
Previous studies have shown that the CD73/A2A pathway reduces disease severity in allogeneic mouse models of GVHD [[11], [12], [13], [14]]. However, the effect of A2A activation in humanised mouse models or in HSCT patients has not been reported. Using a humanised NSG mouse model of GVHD, the current study demonstrated that the A2A agonist CGS 21680 had opposing roles in disease development. CGS 21680 did not affect clinical score or mortality in humanised mice but reduced GVHD severity, as
Acknowledgements
This project was funded by the Faculty of Science, Medicine and Health, University of Wollongong and Molecular Horizons (University of Wollongong). We thank Kathryn Friend (BioLegend) for expert advice and assistance with multiplex assays, and Thomas Guy (University of Wollongong) for expert advice and assistance with flow cytometry. We also thank the technical staff of the Illawarra Health and Medical Research Institute, and the staff of the University of Wollongong Rodent Facility for kind
Disclosure
All authors declare that they have no disclosures.
Author contributions
N.J.G., D.W. and R.S. designed the experiments. N.J.G. and S. R. A. performed the experiments. N. J. G. analysed the data, prepared the figures and wrote the manuscript. S. R. A. co-edited the manuscript. D.W. and R.S. supervised the project, reviewed the data and edited the manuscript.
References (56)
- et al.
Risk factors for acute GVHD and survival after hematopoietic cell transplantation
Blood
(2012) - et al.
Regulatory T cells and natural killer T cells for modulation of GVHD following allogeneic hematopoietic cell transplantation
Blood
(2013) - et al.
The role of danger signals and ectonucleotidases in acute graft-versus-host disease
Hum. Immunol.
(2016) - et al.
The purinergic system in allotransplantation
Am. J. Transplant.
(2014) - et al.
Deficiency of CD73/ecto-5′-nucleotidase in mice enhances acute graft-versus-host disease
Blood
(2012) - et al.
A new Hu-PBL model for the study of human islet alloreactivity based on NOD-scid mice bearing a targeted mutation in the IL-2 receptor gamma chain gene
Clin. Immunol.
(2008) - et al.
Comprehensive analysis of the activation and proliferation kinetics and effector functions of human lymphocytes, and antigen presentation capacity of antigen-presenting cells in xenogeneic graft-versus-host disease
Biol. Blood Marrow Transplant.
(2018) - et al.
Long-term treatment with the P2X7 receptor antagonist Brilliant Blue G reduces liver inflammation in a humanized mouse model of graft-versus-host disease
Cell. Immunol.
(2019) - et al.
Adenosine A2a receptors induce heterologous desensitization of chemokine receptors
Blood
(2006) - et al.
Differences in the induction of induced human CD4+ CD25+ FoxP3+ T-regulatory cells and CD3+ CD8+ CD28− T-suppressor cells subset phenotypes in vitro: comparison of phorbol 12-myristate 13-acetate/ionomycin and phytohemagglutinin stimulation
Transplant. Proc.
(2013)