Graft-versus-host disease (GVHD) is a major complication that occurs following allogeneic haematopoietic stem cell transplantation (HSCT) for the treatment of haematological cancers and other blood-related disorders. GVHD is an inflammatory disorder, where the transplanted donor immune cells can mediate an immune response against the recipient and attack host tissues. Despite over 60 years of research, broad-range immune suppression is still used to prevent or treat GVHD, leading to an increased risk of cancer relapse and infection. Therefore, further insights into the disease mechanisms and development of predictive and prognostic biomarkers are key to improving outcomes and reducing GVHD development following allogeneic HSCT. An important preclinical tool to examine the pathophysiology of GVHD, and to understand the key mechanisms that lead to GVHD development, are preclinical humanised mouse models. Such models of GVHD are now well-established and can provide valuable insights into disease development. This review will focus on models where human peripheral blood mononuclear cells are injected into immune-deficient non-obese diabetic (NOD)-scid-interleukin-2(IL-2)Rγ mutant (NOD-scid-IL2Rγ^null) mice. Humanised mouse models of GVHD can mimic the clinical setting for GVHD development, with disease progression and tissues impacted like that observed in humans. This review will highlight key findings from preclinical humanised mouse models regarding the role of donor human immune cells, the function of cytokines and cell signalling molecules and their impact on specific target tissues and GVHD development. Further, specific therapeutic strategies tested in these preclinical models reveal key molecular pathways important in reducing the burden of GVHD following allogeneic HSCT.

The ATP-gated P2X7 ion channel has emerging roles in amyotrophic lateral sclerosis (ALS) progression. Pharmacological blockade of P2X7 with Brilliant Blue G can ameliorate disease in SOD1^G93A mice, but recent data suggests that this antagonist displays poor penetration of the central nervous system (CNS). Therefore, the current study aimed to determine whether the CNS-penetrant P2X7 antagonist, JNJ-47965567, could ameliorate ALS progression in SOD1^G93A mice. A flow cytometric assay revealed that JNJ-47965567 impaired ATP-induced cation dye uptake in a concentration-dependent manner in murine J774 macrophages. Female and male SOD1^G93A mice were injected intraperitoneally with JNJ-47965567 (30 mg/kg) or 2-(hydroxypropyl)-beta-cyclodextrin (vehicle control) three times a week from disease onset until end stage, when tissues were collected and studied. JNJ-47965567 did not impact weight loss, clinical score, motor (rotarod) coordination or survival compared to control mice. NanoString analysis revealed altered spinal cord gene expression in JNJ-47965567 mice compared to control mice, but such differences were not confirmed by quantitative PCR. Flow cytometric analyses revealed no differences between treatments in the frequencies or activation status of T cell or dendritic cell subsets in lymphoid tissues or in the concentrations of serum cytokines. Notably, serum IL-27, IFNβ and IL-10 were present in relatively high concentrations compared to other cytokines in both groups. In conclusion, JNJ-47965567 administered thrice weekly from disease onset did not alter disease progression or molecular and cellular parameters in SOD1^G93A mice.

Graft-versus-host disease (GVHD) is a severe inflammatory response arising from allogeneic haematopoietic stem cell transplantation. Previous studies revealed that antagonism of the P2X7 receptor with Brilliant Blue G (BBG) reduced liver GVHD but did not alter clinical GVHD in a humanised mouse model. Therefore, the current study aimed to trial a modified injection regime using more frequent dosing of BBG to improve outcomes in this model of GVHD. NOD-scid IL2Rγ^null (NSG) mice were injected intraperitoneal (i.p.) with 10x10⁶ human peripheral blood mononuclear cells (hPBMCs) (day 0), then daily with BBG (50mg/kg) or saline (days 0-10). BBG significantly reduced clinical score, mortality and histological GVHD compared to saline treatment (endpoint). BBG significantly increased proportions of human regulatory T cells (Tregs) and human B cells and reduced serum human interferon-γ compared to saline treatment prior to development of clinical GVHD (day 21). To confirm the therapeutic benefit of P2X7 antagonism, NSG mice were injected i.p. with 10x10⁶ hPBMCs (day 0), then daily with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (300mg/kg) or saline (days 0-10). PPADS increased human Treg proportions compared to saline treatment (day 21), but potential clinical benefits were confounded by increased weight loss with this antagonist. To investigate the role of P2X7 antagonism on Treg survival, hPBMCs were cultured in reduced serum conditions to promote cell death. BBG increased proportions of Tregs (and B cells) compared to saline under these conditions. In conclusion, P2X7 antagonism reduces clinical and histological GVHD in a humanised mouse model corresponding to an increase in human Tregs.

Allogeneic haematopoietic stem cell transplantation is a curative therapy for blood cancers; but results in the development of graft-versus-host disease (GVHD) in up to 70% of recipients. During GVHD, tissue damage results in ATP release into the extracellular compartment activating P2X7 on antigen presenting cells, leading to the release of pro-inflammatory cytokines and subsequent activation of donor T cells. Therefore, the aim of this study was to examine murine (m) P2rx7 and human (h) P2RX7 gene expression in GVHD target organs of humanised mice, and further characterise disease impact in these organs. Methods: NOD-scid IL2Rγ^null (NSG) mice were injected with human peripheral blood mononuclear cells (hu-PBMC-NSG mice) or PBS (control). Leukocytes were assessed by flow cytometry; gene expression was measured by qPCR, and tissue sections examined by histology. Results: Compared to control mice, hu-PBMC-NSG mice had increased mP2rx7 and mP2rx4 expression in the duodenum, ileum and skin. hP2RX7 was expressed in all tissues examined. hu-PBMC-NSG mice also displayed increased mReg3g expression in the duodenum and ileum, despite limited histological gut GVHD. hu-PBMC-NSG mice showed histological evidence of GVHD in the skin, liver and lung. Compared to control mice, hu- PBMC-NSG mice displayed increased ear swelling. Conclusion: Combined this data reveals that P2rx7 is upregulated in gut and skin GVHD and that P2RX7 is present in target tissues of GVHD, corresponding to human leukocyte infiltration. Data also reveals increased mReg3g expression and ear swelling in hu-PBMC-NSG mice, offering new measurements of early stage gut GVHD and skin GVHD, respectively.