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Peer-reviewed

Modelling Graft-Versus-Host Disease in Mice Using Human Peripheral Blood Mononuclear Cells

使用人外周血单核细胞模拟小鼠移植物对抗宿主疾病

MH Manjurul Haque *
DB Dominic A. Boardman *
AL Avery J. Lam
KM Katherine N. MacDonald
LS Lieke Sanderink
QH Qing Huang
VF Vivian C. W. Fung
SI Sabine Ivison
MM Majid Mojibian
ML Megan K. Levings

 (*contributed equally to this work) 发布: 2022年12月05日第12卷第23期 DOI: 10.21769/BioProtoc.4566 浏览次数: 2322

评审: Luis Alberto Sánchez VargasTakashi Nishina

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Abstract

Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies.


Graphical abstract




Keywords: Regulatory T cell (调节性 T 细胞) search Graft-versus-host disease (GvHD) (移植物抗宿主病 (GvHD)) search Hematopoietic stem cell transplantation (HSCT) (造血干细胞移植（HSCT）) search Xenograft (异种移植) search Thymic T-regulatory cells (胸腺 T 调节细胞) search

Background

Hematopoietic stem cell transplantation (HSCT) is a therapeutic strategy used to treat certain immune system disorders as well as various hematological cancers such as myeloma and leukemia (Gschweng et al., 2014). In this procedure, recipients are typically conditioned with irradiation and/or chemotherapy to deplete immune cells, which are, in turn, replaced by an intravenous infusion of hematopoietic stem cells. As the hematopoietic stem cells are often derived from an allogeneic donor, a severe and potentially lethal side effect of HSCT is graft-versus-host disease (GvHD), whereby the patient’s new immune cells recognize the recipient’s cells as foreign and attack and destroy healthy tissues (Ferrara et al., 2009). This process is thought to be driven by reconstituted T cells recognizing allogeneic human leukocyte antigens (HLAs) on recipient cells (Loiseau et al., 2007).


Animal models are critical for testing novel therapeutic strategies that can be used to treat GvHD. Mouse models of GvHD are well established and have significantly advanced this field of research. However, in order to examine how new therapies influence the function of human T cells, models in which mice are reconstituted with human immune cells (humanized mice) are required. Accordingly, significant work has been performed to generate immunodeficient mice that can be reconstituted with human cells from different sources. For example, NOD-scid-IL-2Rγ null (NSG) mice are immunodeficient for B, T, and natural killer (NK) cells and have defective dendritic cells and macrophages, allowing them to be efficiently engrafted with human cells/tissues (Ehx et al., 2018). However, each humanized mouse model has unique properties that must be carefully considered when attempting to answer specific scientific questions. For example, it is well established that following peripheral blood mononuclear cell (PBMC) reconstitution of NSG mice, the primary cells to engraft are T cells, making this model unsuitable for assessing humoral immune responses. Additional factors such as time and cost should also be considered when selecting an appropriate humanized mouse model. This area has been extensively reviewed in Walsh et al. (2017), Adigbli et al. (2020), and Khosravi-Maharlooei et al. (2022).


For modelling the multi-system disorder known as xenoGvHD, we and others have shown that human T cells readily engraft in NSG mice upon intravenous infusion. These human T cells are stimulated in a xenogeneic manner via the recognition of mouse major histocompatibility complex molecules, and as such, the consequential pathology observed in these mice closely mimics human GvHD (King et al., 2009). Although PBMC-reconstituted xenoGvHD mouse models have their limitations, such as the lack of a complete human immune system, they can be used to generate safety data and conduct pre-clinical proof-of-concept data for T cell–directed therapies.


This protocol describes a method for inducing and monitoring xenoGvHD in NSG mice. We provide details on human PBMC preparation, methods to monitor disease progression over time, and an approach to data analysis and reporting. We also provide an example of how this model can be used to demonstrate the therapeutic potential of regulatory T cells (Tregs).

Materials and Reagents

Materials

  1. Sterile 1.5 mL microcentrifuge tubes (Fisher Scientific, catalog number: 229442)

  2. 15 mL Falcon tubes (Corning, catalog number: 14-959-53A)

  3. 50 mL Falcon tubes (Corning, catalog number: 14-432-22)

  4. Heparinized capillaries (Fisherbrand, catalog number: 22-362-566)

  5. 2” × 2” gauze (Fisherbrand, catalog number: 22-362178)

  6. Cotton tipped applicators (Q-tips®)

  7. Petroleum jelly (i.e., Vaseline®)

  8. 27 G needles, one per animal (BD Biosciences, catalog number: 305109)

  9. Appropriate mouse restrainer for blood collection

  10. 70 µm cell strainer (Corning, catalog number: 352350)

  11. 96-well clear V-Bottom polystyrene not treated microplate (Corning, catalog number: 3896)

  12. 0.5 mL insulin syringes (BD Horizon, catalog number: 329461)

  13. 1 mL syringe (BD Horizon, catalog number: 309659)

  14. Weighing bucket (empty 1 mL tip box without the rack or any small box)

  15. Micro test tubes (Bio-Rad, catalog number: 2239391)

  16. Corning® cryogenic vials, internal thread (Corning, catalog number: CLS430488)


Mice

8–16-week-old NOD/SCID/IL-2Rgammanull (NSG) mice (The Jackson Laboratory, Strain: 005557)

Note: Select the sex of mice depending on the experimental question. Both commercially available and house bred mice may be used. We have used mice of either sex and have not observed any differences in the induction of xenoGvHD.


Drugs

Anesthetic, isoflurane (USP 250 mL) (Piramal Critical Care Inc, part number: DVM-102190)


Reagents

  1. Heparin (Sigma, catalog number: H3149-10KU)

  2. 70% ethanol (VWR, catalog number: 89370-078)

  3. Fixation/permeabilization concentrate (eBioscience, catalog number: 00-5123-43)

  4. Fixation/permeabilization diluent (eBioscience, catalog number: 00-5223-56)

  5. 10× permeabilization buffer (eBioscience, catalog number: 00-8333-56)

  6. Counting beads (123count eBeadsTM, Invitrogen, catalog number: 01-1234-42)

  7. DNase I solution (1 mg/mL) (StemcellTM Technologies, catalog number: 07900)

  8. Penicillin–streptomycin (P/S) (GibcoTM, catalog number: 15140122)

  9. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148)

  10. Dithiothreitol (DTT) (ThermoFisher, catalog number: 20290)

  11. EDTA (Sigma-Aldrich, catalog number: 03690)

  12. Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C7657)

  13. Percoll (Sigma-Aldrich, catalog number: P1644)


Media and Buffers

  1. 10× RBC lysis buffer (eBioscience, catalog number: 00-4300-54)

  2. Gibco 1× Dulbecco’s phosphate buffered saline (DPBS) (ThermoFisher, catalog number: 14190)

  3. Gibco 10× Phosphate buffered saline (PBS) (ThermoFisher, catalog number: 70013032)

  4. LymphoprepTM (StemcellTM Technologies, catalog number: 07851)

  5. Immunocult-XF T-cell expansion medium (StemcellTM Technologies, catalog number: 10981)

  6. Gibco fetal bovine serum qualified (FBS) (ThermoFisher, catalog number: 12483020)

  7. Gibco RPMI 1640 medium (ThermoFisher, catalog number: 11875093)

  8. Gibco GlutaMax (ThermoFisher, catalog number: 35050061)

  9. Gibco HEPES (1 M) (ThermoFisher, catalog number: 15630080)


Antibodies

  1. Mouse Fc block (BD Biosciences, catalog number: 553142)

  2. Fixable viability dye (FVD) eFluor 780 (eBioscience, catalog number: 65-0865-18)

  3. Anti-Mouse CD45 (30-F11) AF700 (BD Biosciences, catalog number: 560510)

  4. Anti-Human CD45 (HI30) V500 (BD Biosciences, catalog number: 560777)

  5. Anti-Human CD4 (SK3) BV786 (BD Biosciences, catalog number: 563877)

  6. Anti-Human CD8 (RPA-T8) BV711 (BD Biosciences, catalog number: 563677)

  7. Anti-Human CD3 (UCHT1) BB515 (BD Biosciences, catalog number: 564466)

  8. Anti-Human Foxp3 (236A/E7) PE-Cy7 (eBioscience, catalog number: 25-4777-42)

  9. Anti-Human Helios (22F6) AF488 (BioLegend, catalog number: 137223)

  10. Anti-Human HLA-A2 (BB7.2) APC (Invitrogen, catalog number: 17-9876-42)

Equipment

  1. Type II biosafety cabinet (NuAire, model: LabGard ES NU-540)

  2. Microcentrifuge (Eppendorf, models: 5810R and 5452)

  3. Cell counter (e.g., Nexcelom, model: Cellometer Auto 2000 or alternative)

  4. X-ray irradiator (Rad Source-RS2000 Pro Biological Irradiator; alternate instruments including a gamma irradiator can be used)

  5. Dosimeter (Radcal-2186 Dose Meter; alternative instruments can be used)

  6. Electric shaver (Wahl Professional Animal BravMini+ Pet Trimmer, catalog number: 41590-0437)

  7. Ear notcher (Fine Science Tools, catalog number: 24214-02)

  8. Flow cytometer (BD LSRFortessaTM X-20; alternative instruments can be used)

  9. OHAUSTM NavigatorTM portable balance (Thermo Fisher, catalog number: 01-922-205)

  10. Thermo ScientificTM PrecisionTM general purpose baths (Thermo Fisher, catalog number: TSGP05)

  11. VetFloTM vaporizer single-channel anesthesia system (Kent Scientific Corporation, catalog number: VetFlo-1205SP)

Software

  1. FlowJo (BD Biosciences, 10.8.1)

Procedure

English
中文翻译

文章信息

版权信息

© 2022 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Haque, M., Boardman, D. A., Lam, A. J., MacDonald, K. N., Sanderink, L., Huang, Q., Fung, V. C., Ivison, S., Mojibian, M. and Levings, M. K. (2022). Modelling Graft-Versus-Host Disease in Mice Using Human Peripheral Blood Mononuclear Cells. Bio-protocol 12(23): e4566. DOI: 10.21769/BioProtoc.4566.

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分类

医学 > 发炎

免疫学 > 宿主防御

细胞生物学 > 细胞移植

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