Elsevier

Transplant Immunology

Volume 54, June 2019, Pages 38-46
Transplant Immunology

Increased splenic human CD4+:CD8+ T cell ratios, serum human interferon-γ and intestinal human interleukin-17 are associated with clinical graft-versus-host disease in humanized mice

https://doi.org/10.1016/j.trim.2019.02.003Get rights and content

Highlights

  • GVHD development in humanized mice is variable as most mice develop clinical disease but some develop subclinical disease.

  • Mice with clinical GVHD demonstrate increased splenic human CD4+:CD8+ T cell ratios compared to mice with subclinical GVHD.

  • Mice with clinical GVHD exhibit increased serum human interferon (IFN)-γ compared to mice with subclinical GVHD.

Abstract

Graft-versus-host disease (GVHD) is a frequent complication following allogeneic hematopoietic stem cell transplantation (HSCT) with current therapies limited to general immunosuppression. Humanized mouse models of GVHD are emerging as valuable intermediaries to allow translation of findings from allogeneic mouse models to humans to prevent and treat this disease, but such models require further characterization. In this study, humanized mice were generated by injecting immunodeficient non-obese diabetic severe combined immunodeficiency interleukin (IL)-2 receptor γ common chain null (NSG) mice with human peripheral blood mononuclear cells (hPBMCs). Clinical GVHD development was assessed using established scoring criteria (weight loss, posture, activity, fur texture and skin integrity). Differences between humanized NSG mice that developed clinical or subclinical GVHD were then compared. Both groups of mice demonstrated similar frequencies of human leukocyte engraftment. In contrast, mice that developed clinical GVHD demonstrated increased histological damage compared to mice with subclinical GVHD. Furthermore, mice with clinical GVHD exhibited increases in the splenic human CD4+:CD8+ T cell ratio, serum human interferon (IFN)-γ and intestinal human IL-17 expression compared to mice with subclinical GVHD. These cellular and molecular changes could be used as potential markers of disease progression in this preclinical model. This study also provides further insights into GVHD development which may be relevant to human HSCT recipients.

Introduction

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy for haematological malignancies and other blood disorders. However, graft-versus-host disease (GVHD) occurs in approximately half of the HSCTs conducted annually [1] and leads to a mortality rate of approximately 20% in these patients [2]. GVHD emerges when effector donor T cells mount an immune response against host tissues [3]. GVHD is characterized by three stages. The first stage is release of danger signals from cells damaged by the underlying disease and/or conditioning regimes, followed by CD4+ T cell activation by antigen presenting cells resulting in cytokine release, and finally CD4+ and CD8+ T cell-mediated inflammatory damage [4,5]. This damage can be propagated by the “cytokine storm” in the latter stage, wherein Th1 and Th17 cells release pro-inflammatory cytokines such as interferon (IFN)-γ [4,5] and interleukin (IL)-17 [6,7], respectively, perpetuating a feed forward loop of inflammation. Conversely, regulatory T (Treg) cells can modulate effector T cells to reduce T cell-mediated inflammatory damage in GVHD [8]. Current therapies for GVHD are limited, with the standard therapy being general immunosuppression achieved through steroids, leaving patients susceptible to subsequent infections and/or disease relapse [9]. Therefore, greater understanding of the mechanisms important in GVHD and elucidation of new therapies is essential to improve outcomes in HSCT patients and prevent GVHD.

Current studies into potential therapeutics for GVHD are investigated in allogeneic mouse models before translation to the clinic. However, potentially due to large species differences between humans and mice, therapies that delay or prevent GVHD in allogeneic mouse models often do not translate to the clinic. To address this, previous studies have developed a range of preclinical “humanized” mouse models [10]. The most commonly used of these models is the humanized immunodeficient non-obese diabetic severe combined immunodeficiency IL-2 receptor γ common chain null (NSG) mouse, developed by Shultz et al. [11] wherein mice readily engraft human peripheral blood mononuclear cells (hPBMCs) due to three defects. First, the Scid mutation prevents V(D)J recombination to impair B and T cell development [12]. Second, deletion of the Il2rg gene results in absence of natural killer (NK) cells. Third, a polymorphism in the Sirpa gene, present due to backcrossing onto a NOD background, promotes phagocytic tolerance of xenogeneic leukocytes [13]. Human T cells recognize major histocompatibility complex (MHC) class I and II of NSG mice to cause GVHD in this humanized mouse model [14], demonstrating that human T cell responses can be investigated in vivo to better understand this disease in a preclinical setting.

The humanized NSG model of GVHD is emerging as a valuable intermediate to allow translation of findings identified in allogeneic mouse models to human clinical trials. For example, based on an earlier study in an allogeneic mouse model of GVHD [15], King, et al. [14] demonstrated tumour necrosis factor (TNF)-α blockade could reduce GVHD severity in this humanized mouse model, and etanercept, an anti-TNF-α monoclonal antibody (mAb) has subsequently shown efficacy in clinical trials [16]. More recently, Burger et al. [17] demonstrated an anti-CCR5 mAb (PRO 140) could reduce GVHD in humanized NSG mice, with this mAb now being investigated in clinical trials [18]. Finally, the discovery of Treg cells, and their therapeutic potential in GVHD has progressed from allogeneic mouse models [8], to humanized NSG models [19,20], and finally to clinical trials [21]. Moreover, this model has helped elucidate the action of post-transplant cyclophosphamide on Treg cells in GVHD development [22]. Therefore, the humanized NSG model offers a valid preclinical model to test therapies for translation to the clinic. Although this mouse model has offered numerous advancements, the characterization of GVHD in these mice has not been adequately described.

Section snippets

Objective

The current study aimed to investigate the characteristics of GVHD that were observed in humanized NSG mice injected with human PBMCs that developed clinical disease compared to those mice that displayed subclinical GVHD. This study demonstrated that all humanized NSG mice had similar engraftment of human leukocytes, which were predominantly T cells. In contrast, humanized NSG mice with clinical GVHD demonstrated greater splenic CD4+:CD8+ T cells ratios, serum human IFN-γ concentrations and

Antibodies for flow cytometry

Fluorescein isothiocyanate (FITC)-conjugated mouse anti-hCD4 (clone: RPA-T4), and mouse anti-hCD45 (clone: HI30); R-phycoerythrin (PE)-conjugated mouse anti-hCD3 (clone: UCHT1) and mouse anti-hCD8 (clone: RPA-T8); peridinin chlorophyll protein (PerCP)-Cy5.5 conjugated mouse anti-hCD4 (clone: L200) and rat anti-mCD45 (clone: 30-F11); and allophycocyanin (APC)-conjugated mouse anti-hCD3 (clone: UCHT1) and mouse anti-hCD19 (clone: HIB19) mAb were obtained from BD (San Jose, CA, USA).

Mice

All mouse

Clinical GVHD development varies in humanized NSG mice

NSG mice, injected i.p. with either saline (control) or hPBMCs, were monitored for physical manifestations of GVHD over 8 weeks. After 4 weeks, humanized NSG mice with GVHD started to exhibit clinical signs of disease (Fig. 1A). By end-point it became apparent that hPBMC-injected mice segregated into one of two groups based on clinical score (Table 1); one group with scores ≥3 (clinical GVHD) and another group with scores <3 (subclinical GVHD) (Fig. 1A). Mice with clinical scores ≥3 exhibited

Discussion

Humanized NSG mouse models of GVHD are used as preclinical models to test potential therapeutics [10], however these models have not been fully characterized. The current study compared humanized NSG mice with subclinical and clinical GVHD, which paralleled the degree of histological disease. Notably, mice with clinical GVHD exhibited greater splenic hCD4+:hCD8+ T cell ratios, serum hIFN-γ concentrations and intestinal hIL-17 expression than mice with subclinical GVHD. However, it should be

Acknowledgements

This project was funded by the Illawarra Health and Medical Research Institute and Molecular Horizons (University of Wollongong) and the AMP Tomorrow Fund. We would like to thank the technical staff of the Illawarra Health and Medical Research Institute, and the animal house staff at the Westmead Animal Facility and the University of Wollongong for assistance.

Conflict of interest

The authors declare that they have no conflicts of interest.

Author contributions

N. J. G., L. B., S. R. A., S. I. A., R. S. and D. W. designed and performed the experiments. N. J. G., L. B., S. R. A. analyzed the data. N. J. G. prepared the figures and wrote the manuscript. L. B. and S. R. A reviewed the manuscript. S. I. A. provided mice and reviewed the manuscript. R. S. and D. W. supervised the project, reviewed the data and edited the manuscript.

Reference list (49)

  • T. Cao et al.

    Ex vivo expanded human CD4+CD25+Foxp3+ regulatory T cells prevent lethal xenogenic graft versus host disease (GVHD)

    Cell. Immunol.

    (2009)
  • P. Trzonkowski et al.

    First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+CD127− T regulatory cells

    Clin. Immunol.

    (2009)
  • G. Vlad et al.

    Suppression of xenogeneic graft-versus-host disease by treatment with immunoglobulin-like transcript 3-Fc

    Hum. Immunol.

    (2009)
  • Y. Kawasaki 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)
  • S. Abraham et al.

    IL-10 exacerbates xenogeneic GVHD by inducing massive human T cell expansion

    Clin. Immunol.

    (2015)
  • K.L. Hippen et al.

    Blocking IL-21 signaling ameliorates xenogeneic GVHD induced by human lymphocytes

    Blood

    (2012)
  • V.H. Nguyen et al.

    In vivo dynamics of regulatory T-cell trafficking and survival predict effective strategies to control graft-versus-host disease following allogeneic transplantation

    Blood

    (2007)
  • M. Miyara et al.

    Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor

    Immunity

    (2009)
  • A. Varelias et al.

    Acute graft-versus-host disease is regulated by an IL-17–sensitive microbiome

    Blood

    (2017)
  • S.Z. Pavletic et al.

    Are we making progress in GVHD prophylaxis and treatment?

    ASH Educ. Program Book

    (2012)
  • R.E. Billingham

    The biology of graft-versus-host reactions

    Harvey Lect.

    (1965)
  • M. Edinger et al.

    CD4+ CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation

    Nat. Med.

    (2003)
  • J.J. Auletta et al.

    Bone marrow transplantation: new approaches to immunosuppression and management of acute graft-versus-host disease

    Curr. Opin. Pediatr.

    (2009)
  • L.D. Shultz et al.

    Humanized mice in translational biomedical research

    Nat. Rev. Immunol.

    (2007)
  • Cited by (15)

    View all citing articles on Scopus
    View full text