Insights into mechanisms of graft-versus-host disease through humanised mouse models

Abstract

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-IL2Rγnull) 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.

Keywords: Allogeneic haematopoietic stem cell transplantation; Antigen presenting cell; Graft-versus-host disease; Inflammation; Lymphocyte; Xenogeneic graft-versus-host disease.

Figure 1. Humanised mouse models of graft-versus-host disease

To establish a model of GVHD, non-pre-conditioned or pre-conditioned (1–3.5 Gy radiation) NOD.Cg-Prkdc^scidIl2rg^tm1Wjl (NSG), NOD.Cg-Prkdc^scidIl2rg^tm1Sug (NOG) or NOD.Prkdc^scidIl2rg^null (NPG) mice are injected intraperitoneally (i.p.) or intravenously (i.v.) with 1 × 10⁵ – 2 × 10⁷ hPBMCs (Day 0). Mice begin to develop clinical signs of GVHD from 3 to 4 weeks post-hPBMC injection. Clinical signs of GVHD include weight loss, fur ruffling, fur loss, hunching, lethargy and ear swelling. Disease modifying reagents can be administered at selected time points as desired. At experimental or humane endpoints, tissues such as blood, spleen, liver, lung, gastrointestinal tract, bone marrow, skin, ear and serum can be collected and analysed. Techniques for analysis of tissues include, but are not limited to, flow cytometry, histology (including immunohistochemistry), enzyme-linked immunosorbent assay (ELISA) (and other immunosorbent assays and biochemical assays) and quantitative real-time polymerase chain reaction (qPCR) (and other gene expression assays).

Figure 2. Cellular mechanisms in graft-versus-host disease

Immunodeficient mice are humanised with hPBMCs. Both mouse and human APCs become activated through interaction with either endogenous or exogenous antigen (Ag). Pre-conditioning regimens lead to the release of cytokines. CD4⁺ and CD8⁺ T cells are then stimulated by activated mouse or human APCs. CD4⁺ and CD8⁺ T cells proliferate and differentiate to mediate tissue destruction. Allogeneic mouse studies suggest that these processes may involve the Fas/FasL with pro-inflammatory cytokine release and perforin/granzyme B pathways, respectively. Destruction of mouse tissues leads to the further release of cross-reactive pro-inflammatory cytokines, including IFN-γ and IL-17, which creates an inflammatory immune response via a positive feedback loop. Tregs suppress the proliferation of CD4⁺ and CD8⁺ T cells and attenuate GVHD severity. MSCs and MDSCs suppress T-cell differentiation and proliferation whilst simultaneously stimulating the generation of Tregs. APCs may also have tolerogenic roles to suppress GVHD development (not shown).