The A2A receptor agonist CGS 21680 has beneficial and adverse effects on disease development in a humanised mouse model of graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative method for blood cancers and other blood disorders, but is limited by the development of graft-versus-host disease (GVHD). GVHD results in inflammatory damage to the host liver, gastrointestinal tract and skin, resulting in high rates of morbidity and mortality in HSCT recipients. Activation of the A2A receptor has been previously demonstrated to reduce disease in allogeneic mouse models of GVHD. This study aimed to investigate the effect of A2A activation on disease development in a humanised mouse model of GVHD. Immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (IL)-2 receptor γnull (NSG) mice injected with human (h) peripheral blood mononuclear cells (hPBMCs), were treated with either the A2A agonist CGS 21680 or control vehicle. Contrary to the beneficial effect of A2A activation in allogeneic mouse models, CGS 21680 increased weight loss, and failed to reduce the clinical score or increase survival in this humanised mouse model of GVHD. Moreover, CGS 21680 reduced T regulatory cells and increased serum human IL-6 concentrations. Conversely, CGS 21680 reduced serum human tumour necrosis factor (TNF)-α concentrations and leukocyte infiltration into the liver, indicating that A2A activation can, in part, reduce molecular and histological GVHD in this model. Notably, CGS 21680 also prevented healthy weight gain in NSG mice not engrafted with hPBMCs suggesting that this compound may be suppressing appetite or metabolism. Therefore, the potential benefits of A2A activation in reducing GVHD in HSCT recipients may be limited and confounded by adverse impacts on weight, decreased T regulatory cell frequency and increased IL-6 production.

Altered donor P2X7 activity in human leukocytes correlates with P2RX7 genotype but does not affect the development of graft-versus-host disease in humanised mice.

Graft-versus-host disease (GVHD) is a life-threatening consequence of allogeneic haematopoietic stem cell transplantation, a curative therapy for haematological malignancies. The ATP-gated P2X7 receptor channel is implicated in the development of GVHD. P2X7 activity on human leukocytes can be influenced by gain-of-function (GOF) and loss-of-function (LOF) single nucleotide polymorphisms (SNPs) in the P2RX7 gene. In this study, the P2RX7 gene was sequenced in 25 human donors and the P2X7 activity on subsets of peripheral blood T cells, natural killer (NK) cells and monocytes was measured using an ATP-induced dye uptake assay. GOF and LOF SNPs representing 10 of the 17 known P2RX7 haplotypes were identified, and correlated with P2X7 activity on all leukocyte subsets investigated. Notably, invariant (i) NK T cells displayed the highest P2X7 activity amongst all cell types studied. To determine if donor P2X7 activity influenced the development of GVHD, immunodeficient NOD-SCID-IL2Rγnull (NSG) mice were injected with human peripheral blood mononuclear cells isolated from donors of either GOF (hP2X7GOF mice) or LOF (hP2X7LOF mice) P2RX7 genotype. Both hP2X7GOF and hP2X7LOF mice demonstrated similar human leukocyte engraftment, and showed comparable weight loss, GVHD clinical score and overall survival. Donor P2X7 activity did not affect human leukocyte infiltration or GVHD-mediated tissue damage, or the relative expression of human P2X7 or human interferon-γ (hIFNγ) in tissues. Finally, hP2X7GOF and hP2X7LOF mice demonstrated similar concentrations of serum hIFNγ. This study demonstrates that P2X7 activity correlates with donor P2RX7 genotype on human leukocyte subsets important in GVHD development, but does not affect GVHD development in a humanised mouse model of this disease.

Long-term treatment with the P2X7 receptor antagonist Brilliant Blue G reduces liver inflammation in a humanized mouse model of graft-versus-host disease.

Allogeneic haematopoietic stem cell transplantation (HSCT) is a frequent curative therapy for numerous haematological malignancies. However, HSCT is limited by the occurrence of graft-versus-host disease (GVHD), with current therapies restricted to general immunosuppression. Activation of the P2X7 receptor by extracellular adenosine triphosphate (ATP) causes inflammation and tissue damage in GVHD. Short-term pharmacological blockade of P2X7 has been shown to reduce clinical disease and/or reduce inflammatory markers in allogeneic and humanized mouse models of GVHD. The current study demonstrates that long-term P2X7 blockade by intra-peritoneal injection of Brilliant Blue G (BBG) thrice weekly for up to 10 weeks did not impact human (h) peripheral blood mononuclear cell (PBMC) engraftment, predominantly T cells, in blood at 3 weeks post-hPBMC injection or in spleens at end-point in humanized mice. Histological analysis demonstrated long-term BBG treatment reduced leukocyte infiltration in the livers of humanized mice. Immunohistochemical analysis demonstrated that BBG treatment reduced liver apoptosis. Long-term BBG treatment did not alter clinical disease, mRNA expression of pro-inflammatory markers in tissues or serum human interferon (IFN)-γ concentrations. Therefore, this study demonstrates that P2X7 activation plays a role in GVHD pathogenesis in the livers of humanized mice, supporting a role for this receptor in GVHD development in HSCT recipients.

The P2X7 receptor antagonist Brilliant Blue G reduces serum human interferon-γ in a humanized mouse model of graft-versus-host disease.

Graft-versus-host disease (GVHD) remains a major problem after allogeneic haematopoietic stem cell transplantation, a curative therapy for haematological malignancies. Previous studies have demonstrated a role for the adenosine triphosphate (ATP)-gated P2X7 receptor channel in allogeneic mouse models of GVHD. In this study, injection of human peripheral blood mononuclear cells (PBMCs) into immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (NOD-SCID-IL)-2Rγnull (NSG) mice established a humanized mouse model of GVHD. This model was used to study the effect of P2X7 blockade in this disease. From five weeks post-PBMC injection, humanized mice exhibited clinical signs and histopathology characteristic of GVHD. The P2X7 antagonist, Brilliant Blue G (BBG), blocked ATP-induced cation uptake into both murine and human cells in vitro. Injection of BBG (50 mg/kg) into NSG mice did not affect engraftment of human leucocytes (predominantly T cells), or the clinical score and survival of mice. In contrast, BBG injection reduced circulating human interferon (IFN)-γ significantly, which was produced by human CD4+ and CD8+ T cells. BBG also reduced human T cell infiltration and apoptosis in target organs of GVHD. In conclusion, the P2X7 antagonist BBG reduced circulating IFN-γ in a humanized mouse model of GVHD supporting a potential role for P2X7 to alter the pathology of this disease in humans.