Debbie Watson

The P2X7 receptor (or P2X7) is an ATP-gated ion channel with important roles in the immune and other body systems. Much has been garnered about P2X7 using the first published anti-human P2X7 monoclonal antibody (mAb) (clone L4). This mAb can be used to impair P2X7 channel and pore activity and events downstream of P2X7 activation in vitro and in vivo. Immunolabelling with this mAb has been used in flow and mass cytometry, confocal microscopy and immunohistochemistry to help establish the presence of P2X7 on human leukocytes and other primary cell types, as well as various malignant cell lines. This mAb has been applied to immunoprecipitate P2X7 to identify co-associated molecules to help uncover novel roles of the receptor such as phagocytosis. Recently, this mAb has been shown to mediate complement-dependent cytotoxicity, offering a new avenue of limiting P2X7-mediated processes by depleting P2X7 ⁺ cells. Finally, this mAb has been used to identify and characterise polymorphic and other variants of P2X7. This article will discuss the origins and various applications of the anti-P2X7 mAb (clone L4). As an early user of this mAb and pioneer of other purinergic technologies, this article is a tribute to the late Francesco Di Virgilio.

P2X7 is an adenosine 5′-triphosphate (ATP)-gated ion channel. CD39 (ectonucleotidase triphosphate diphosphohydrolase-1) hydrolyses ATP to reduce its extracellular concentration to limit P2X7 activation. Both P2X7 and CD39 are present on leukocytes. Ficoll-Paque density gradient centrifugation is widely used to isolate human peripheral blood mononuclear cells (PBMCs), yet the extent to which other density gradient centrifugation methods influence the expression of cell surface molecules remains unclear. Using mass cytometry, this study compared the proportions of mononuclear leukocyte subsets and the relative amount of cell surface P2X7 and CD39 detected on these cells in paired, bar-coded cryopreserved human PBMC samples isolated by Ficoll-Paque or SepMate Tube density gradient centrifugation. Both techniques yielded similar proportions of mononuclear leukocyte subsets. P2X7 and CD39 were detected across all cell subsets, with the relative amount of P2X7 or CD39 comparable between separation methods. Relatively minor but statistically significant differences were observed for some populations. P2X7 expression was higher on CD3⁺, CD4⁺, and conventional CD4⁺ T cells, and naïve B cells, and lower on myeloid dendritic cells, while CD39 expression was lower on regulatory T cells in SepMate Tube samples compared to Ficoll-Paque samples. Overall, Ficoll-Paque and SepMate Tube density gradient centrifugation yield comparable results within PBMC samples, supporting the use of either method in studies examining immune phenotypes including the purinergic molecules P2X7 and CD39.

Allogeneic haematopoietic stem cell transplantation (alloHSCT) is a curative treatment for haematological malignancies. AlloHSCT aims to generate graft-versus-leukaemia immunity, where donor T cells eliminate residual malignant cells. However, graft-versus-host disease (GVHD), where donor T cells attack recipient tissues, is a common and often fatal side effect. Post-transplant cyclophosphamide (PTCy) can reduce GVHD, but the cellular mechanisms through which this occurs are not fully understood, and high doses may be associated with toxicity. This study aimed to determine whether lower doses of PTCy can reduce GVHD and to examine the effects of PTCy doses on human (h) immune cell subsets, T cell exhaustion, and histological GVHD in a humanised mouse model. NOD-scid-IL2Rγnull mice were injected with 2 × 107 human peripheral blood mononuclear cells on day 0, cyclophosphamide (10, 25 or 33 mg/kg) or control diluent on days 3 and 4 and monitored for GVHD development at early and late time points. Low-dose PTCy (10 mg/kg) abrogated clinical signs of GVHD with comparable efficacy to high-dose PTCy (33 mg/kg), delaying GVHD onset and prolonging mouse survival. Proportions of hPD-1+ hCD4+ and hPD-1+hCD8+ T cells were increased with low-dose PTCy but not higher doses, while hPD-1+ hTreg proportions were increased by all PTCy doses. Exhausted hPD-1+hLAG3+hCD8+ T cell proportions were increased with high-dose PTCy, but not lower doses. This study indicates that low-dose PTCy reduces GVHD with similar efficacy to that of high-dose PTCy, but this appears to be associated with differing cellular mechanisms of action.

Humanized mouse models of graft-versus-host disease (GVHD) primarily focus on treatments to prevent disease progression. However, there is little investigation into whether graft-versus-leukemia (GVL) immunity is retained following these treatments for GVHD. Here, we describe the methods to generate a humanized mouse model to study GVL immunity against firefly luciferase (luc) expressing human K562 chronic myeloid leukemia cells in humanized NOD-scid-IL2Rγ^null mice. This chapter provides an overview of human peripheral blood mononuclear cell isolation, culturing of K562-luc chronic myeloid leukemia cells, injection of these cells into immunodeficient mice, monitoring for signs of disease, tracking of leukemia using an in vivo imaging system, and assessment of human cell engraftment and GVL immunity using flow cytometry.

Allogeneic hematopoietic stem cell transplantation (HSCT) is used to treat malignant and non-malignant blood disorders, but its efficacy is limited by the development of graft-versus-host disease (GVHD). Thus, better understanding and new biomarkers and therapies are required to combat this disease. Xenogeneic animal models of GVHD help to address these gaps and to translate findings from allogeneic animal models of GVHD and other laboratory findings to human allogeneic HSCT recipients. This chapter discusses different humanized mouse models used to study GVHD. Further, this chapter introduces a humanized rat model of GVHD, as well as other xenogeneic models of GVHD in which immune-deficient mice receive blood cells from dogs, cows, or horses. Collectively, this chapter serves as a brief account of xenogeneic animal models of GVHD.

Humanized mouse models of graft-versus-host disease (GVHD), involving the injection of human immune cells into immunodeficient mice, are firmly established and offer avenues for exploring pathways implicated in GVHD development and for testing therapeutics to prevent or treat this disease. NOD-scid-IL-2Rγ^null (NSG) mice are a strain of immunodeficient mice commonly used to study GVHD. This chapter details methods relating to this model including human immune cell isolation, the injection of these human immune cells into nonirradiated NSG mice, monitoring the mice for signs of clinical GVHD (including weight loss), immunolabeling with anti-human and anti-mouse monoclonal antibodies of leukocytes isolated from the spleens, livers, and lungs of humanized mice, evaluating human cell engraftment and human and mouse immune cell subsets via flow cytometry and measuring serum cytokine concentrations using a multiplexed flow cytometric assay.

P2X7 is an extracellular adenosine 5′‐triphosphate (ATP)‐gated cation channel that plays various roles in inflammation and immunity. P2X7 is present on peripheral blood monocytes, dendritic cells (DCs), and innate and adaptive lymphocytes. The anti‐human P2X7 monoclonal antibody (mAb; clone L4), used for immunolabelling P2X7 or blocking P2X7 activity, is a murine IgG2_b antibody, but its ability to mediate complement‐dependent cytotoxicity (CDC) is unknown. In this study the functionality of this mAb was confirmed by inhibition of ATP‐induced Ca²⁺ responses in HEK‐293 cells expressing P2X7 (HEK‐P2X7). Spectrophotometric measurements of lactate dehydrogenase release demonstrated that the anti‐P2X7 mAb mediated CDC in HEK‐P2X7 but not HEK‐293 cells. Further, flow cytometric measurements of the viability dye, 7‐aminoactinomycin D, showed that this mAb mediated CDC in human RPMI 8226 but not mouse J774 cells. Immunolabelling with this mAb and flow cytometry revealed that relative amounts of cell surface P2X7 varied between human peripheral blood leukocytes. As such, the anti‐P2X7 mAb preferentially mediated CDC of leukocytes that displayed relatively high cell surface P2X7, namely monocytes, DCs, natural killer T cells, myeloid‐derived suppressor cells, and T helper 17 cells. Together, this data highlights a novel approach to target cellular P2X7 and to limit unwanted P2X7 functions.

Objectives: Graft‐versus‐host disease (GVHD) is an inflammatory disorder that arises following allogeneic haematopoietic stem cell transplantation. P2X7 is an extracellular ATP‐gated cation channel present on immune cells. P2X7 blockade with small molecule inhibitors impairs GVHD development in a humanised mouse model. This study investigated whether adeno‐associated viral (AAV) vectors encoding nanobodies (Nbs) that block mouse P2X7 (mP2X7) or both mP2X7 and human P2X7 (m/hP2X7) impair GVHD development in this model. Methods: On Day −21, NOD.Cg‐ Prkdc ^scid Il2rg ^tm1Wjl /SzJ (NSG) mice were injected intramuscularly with 10 × 10 ¹⁰ viral genomes encoding either green fluorescent protein (GFP), an anti‐mP2X7 Nb or an anti‐m/hP2X7 Nb, or with saline. On Day 0, mice were euthanised or injected intraperitoneally with 10 × 10 ⁶ human peripheral blood mononuclear cells and monitored thrice weekly for signs of GVHD until the experiment or disease endpoint. Results: The anti‐m/hP2X7 and anti‐mP2X7 Nbs reduced clinical GVHD and time to disease onset, as well as liver and lung GVHD. Both Nbs reduced liver human T helper (Th)17 cells. Sera collected at Day 0 and disease endpoint from treated mice, but not from control mice, completely blocked P2X7 activity in human RPMI 8226 and/or murine J774 cells, confirming circulating anti‐P2X7 Nbs in mice from Day 0 to disease endpoint. Conclusion: This study indicates that P2X7 blockade with an anti‐m/hP2X7 and to a lesser extent an anti‐mP2X7 Nb reduces GVHD progression in humanised mice. This supports the future testing of these P2X7 biologics as a prophylactic therapy for GVHD.

Allogeneic haematopoietic stem cell transplantation (HSCT) leads to the establishment of graft-versus-leukaemia (GVL) immunity, but in many cases also results in the development of graft-versus-host disease (GVHD). This study aimed to determine if P2X7 antagonism using Brilliant Blue G (BBG) could improve the beneficial effects of post-transplant cyclophosphamide (PTCy) in a humanised mouse model of GVHD, without comprising GVL immunity. NOD.Cg-Prkdc^scid Il2rg^tm1Wjl (NSG) mice were injected with human peripheral blood mononuclear cells (PBMCs) (Day 0), then with cyclophosphamide (33 mg/kg) on Days 3 and 4, and with BBG (50 mg/kg) (or saline) on Days 0–10. PTCy with BBG reduced clinical GVHD development like that of PTCy alone. However, histological analysis revealed that the combined treatment reduced liver GVHD to a greater extent than PTCy alone. Flow cytometric analyses revealed that this reduction in liver GVHD by PTCy with BBG corresponded to an increase in human splenic CD39⁺ Tregs and a decrease in human serum interferon-γ concentrations. In additional experiments, humanised NSG mice, following combined treatment, were injected with human THP-1 acute myeloid leukaemia cells on Day 14. Flow cytometric analyses of liver CD33⁺ THP-1 cells showed that PTCy with BBG did not mitigate GVL immunity. In summary, PTCy combined with BBG can reduce GVHD without compromising GVL immunity. Future studies investigating P2X7 antagonism in combination with PTCy may lead to the development of novel treatments that more effectively reduce GVHD in allogeneic HSCT patients without promoting leukaemia relapse.

Objectives: Donor haematopoietic stem cell transplantation treats leukaemia by inducing graft‐versus‐leukaemia (GVL) immunity. However, this benefit is often mitigated by graft‐versus‐host disease (GVHD), which is reduced by post‐transplant cyclophosphamide (PTCy) alone or combined with tocilizumab (TOC) in humanised mice. This study established a preclinical humanised mouse model of GVL and investigated whether PTCy alone or combined with TOC impacts GVL immunity.Methods: NOD‐scid‐IL2Rγ^null mice were injected with 2 × 10⁷ human peripheral blood mononuclear cells (hPBMCs) on day 0 and with 1 × 10⁶ THP‐1 acute myeloid leukaemia cells on day 14. In subsequent experiments, mice were also injected with PTCy (33 mg kg⁻¹) or Dulbecco's phosphate buffered saline (PBS) on days 3 and 4, alone or combined with TOC or control antibody (25 mg kg⁻¹) twice weekly for 28 days. Clinical signs of disease were monitored until day 42.Results: Mice with hPBMCs from three different donors and THP‐1 cells showed similar survival, clinical score and weight loss. hCD33⁺ leukaemia cells were minimal in mice reconstituted with hPBMCs from two donors but present in mice with hPBMCs from a third donor, suggesting donor‐specific GVL responses. hPBMC‐injected mice treated with PTCy alone or combined with TOC (PTCy + TOC) demonstrated prolonged survival compared to control mice. PTCy alone and PTCy + TOC‐treated mice with hPBMCs showed minimal hepatic hCD33⁺ leukaemia cells, indicating sustained GVL immunity. Further, the combination of PTCy + TOC reduced histological damage in the lung and liver.Conclusion: Collectively, this research demonstrates that PTCy alone or combined with TOC impairs GVHD without compromising GVL immunity.