Animal Models of Acute and Chronic Graft‐Versus‐Host Disease

Frances Hakim1, Daniel H. Fowler1, Gene M. Shearer1, Ronald E. Gress1

1 National Cancer Institute NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 4.3
DOI:  10.1002/0471142735.im0403s27
Online Posting Date:  May, 2001
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Abstract

Graft‐versus‐host disease (GVHD) represents a special situation in transplantation immunology in which immunocompetent donor cells are engrafted into recipients that are incapable of rejecting them due to tolerance, immaturity, or radiation‐ or chemotherapy‐induced immune deficiency. Donor T cells encountering allogeneic stimulators become activated, secrete cytokines, proliferate, and differentiate into effectors; this in vivo immune response is known as the graft‐versus‐host reaction (GVHR). The systemic effects of this initial donor anti‐host reaction comprise a multiorgan syndrome, graft‐versus‐host disease (GVHD). Murine GVHD experiments have been utilized to model the clinical disorders of acute and chronic GVHD (AGVHD and CGVHD) that occur after allogeneic bone marrow transplantation, and also to study T cell regulation, induction of tolerance, and autoimmune diseases. Presented in this unit are methods for generating and assessing both AGVHD and CGVHD in mice. While the two syndromes differ markedly in immunopathogenesis, both can be induced by the two main methods presented: transfer of allogenic donor lymphocytes and stem cells into irradiated hosts, and transfer of parental strain lymphocytes and stem cells into unirradiated, immune‐competent F1 strain hosts. Several endpoints of AGVHD and CGVHD should be evaluated in experimental mice, with comparisons made to the syngeneic transplant control or the T cell‐depleted allogeneic control. To this end, protocols are provided for the assessment of survival rates, weight loss, chimerism, donor‐host cytotoxicity, and cytokine and proliferative responses to mitogenic or allogeneic stimuli. Histopathology and assays of B cell immune function are also described for evaluation of the pathogenesis of GVHD.

     
 
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Table of Contents

  • Basic Protocol 1: Induction of Graft‐Versus‐Host Disease in Irradiated Hosts
  • Basic Protocol 2: Induction of Graft‐Versus‐Host Disease in Unirradiated, Immunocompetent Adult F1 Host Mice
  • Support Protocol 1: Assessment of Acute and Chronic GVHD Based on Survival Outcome
  • Support Protocol 2: Assessment of Acute GVHD Based on Weight Loss
  • Support Protocol 3: Assessment of Donor/Host Chimerism in Mice with Acute or Chronic GVHD by Flow Cytometry
  • Support Protocol 4: Assessment of Acute GVHD by Measurement of Donor Anti‐Host Cytotoxicity
  • Support Protocol 5: Assessment of Cytokine Production and Proliferative Responses in Acute and Chronic GVHD
  • Support Protocol 6: Histopathological Assessment of Acute and Chronic GVHD
  • Support Protocol 7: Assessment of Serum Antibody Levels (Humoral Immunity) in Chronic GVHD
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of Graft‐Versus‐Host Disease in Irradiated Hosts

  Materials
  • Appropriate allogeneic donor and host mice (see Table 4.3.1)
  • 70% ethanol
  • Hanks balanced salt solution ( appendix 2A) containing 5 mM HEPES (HBSS/HEPES), sterile
  • Anti–Thy 1.2 antibody (clone HO‐13.4; ATCC) and complement (C′ rabbit low‐tox‐M, Accurate Chemical), for ex vivo depletion of T cells in donor bone marrow (optional)
  • Heparin (optional)
  • Anti–αβ TCR antibodies, anti‐CD4 (clone GK1.5; ATCC) or anti‐CD8 (clone 2.43; ATCC) antibodies, or anti‐NK1.1 or anti‐asialo‐GM1 antibodies (optional, for in vivo T cell depletion of host animals; see Table 97.80.4711 for further listings of T cell–specific antibodies)
  • Sets of dissection tools containing fine scissors and forceps, autoclaved
  • Petri dishes
  • Fine mesh (autoclaved) or filtration cups (Falcon sterile cell strainer)
  • 3‐cc syringes with Luer‐Lok tip
  • 27‐G needles
  • Cesium source for γ‐irradiation of whole animals (e.g., Gammacell 40 irradiator, MDS Nordion)
  • Heat lamp
  • Animal restrainer (for injections)
  • Additional reagents and equipment for animal euthanasia (unit 1.8), removal of spleen and lymph nodes (unit 1.9), preparation of bone marrow cells (unit 6.4), preparation of cell suspensions (unit 3.1), depletion of T cells and T cell subsets (unit 3.4; optional), flow cytometry (units 5.3 & 5.4; optional), T cell quantitation by limiting dilution (unit 3.15; optional), cell counting ( 3.NaN), intravenous injection (unit 1.6), and in vivo T cell depletion (unit 4.1; optional)
NOTE: Keep cells on ice throughout the preparation procedures.

Basic Protocol 2: Induction of Graft‐Versus‐Host Disease in Unirradiated, Immunocompetent Adult F1 Host Mice

  • Appropriate allogeneic donor (parental) and host (F 1) mice (see Table 4.3.2)
  • Interleukin 2 (IL‐2; Chiron)
  • Interleukin 4 (IL‐4; Peprotech)

Support Protocol 1: Assessment of Acute and Chronic GVHD Based on Survival Outcome

  Materials
  • Mice with AGVHD or CGVHD (see protocol 1Basic Protocols 1 and protocol 22)
  • Untreated donor‐ and host‐strain mice
  • Antibody to block Fc receptor binding (clone 24G‐2, Pharmingen), diluted 1/10 in recipeFACS buffer
  • FITC‐labeled antibodies against CD4, CD8, and B220 (e.g., Pharmingen or Caltag)
  • Biotin‐labeled antibodies against host MHC class I and class II antigens (Pharmingen)
  • recipeFACS buffer (see recipe)
  • Streptavidin‐phycoerythrin (streptavidin‐PE; Caltag)
  • 20 µg/ml propidium iodide (Sigma) or 7‐aminoactinomycin D (Sigma) in recipeFACS buffer
  • Additional reagents and equipment for ammonium chloride lysis of erythrocytes (unit 3.1), cell counting with trypan blue exclusion ( appendix 3B), and flow cytometry (units 5.3 & 5.4)

Support Protocol 2: Assessment of Acute GVHD Based on Weight Loss

  Materials
  • Mice with P→F 1 AGVHD (see protocol 1)
  • Donor‐strain mice
  • F 1 host‐strain mice, untreated or injected with F 1 spleen cells
  • HBSS ( appendix 2A)
  • Complete RPMI‐10 ( appendix 2A)
  • Triton X‐100
  • U‐bottom 96‐well microtiter plate
  • γ scintillation counter
  • Additional reagents and equipment for counting cells ( appendix 3A) and measuring cytotoxic T lymphocyte activity (unit 3.11)
NOTE: All cell culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator.

Support Protocol 3: Assessment of Donor/Host Chimerism in Mice with Acute or Chronic GVHD by Flow Cytometry

  Materials
  • Mice with CGVHD (see protocol 2)
  • Untreated donor‐ and host‐strain mice
  • Complete RPMI‐10 ( appendix 2A)
  • Mitomycin C (optional)
  • Concanavalin A (Con A; Sigma)
  • Anti–mouse CD25 MAb (clone 7D4; Pharmingen)
  • Lipopolysaccharides (LPS; Sigma)
  • [3H]Thymidine
  • Cesium source for γ‐irradiation of whole animals capable of delivering 2500 rad
  • 24‐well and flat‐bottomed 96‐well microtiter plates
  • Additional equipment and reagents for counting cells ( appendix 3A), IL‐2 ELISA or CTLL bioassay (unit 6.3), blocking of cellular division of stimulator cells (unit 3.12), and cell labeling and harvesting ( appendix 3D)
NOTE: All cell culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator.

Support Protocol 4: Assessment of Acute GVHD by Measurement of Donor Anti‐Host Cytotoxicity

  Materials
  • recipeMethylated bovine serum albumin (MBSA; see recipe)
  • recipeDulbecco's PBS/Tween (DPBS/Tween; see recipe)
  • recipe10 µg/ml denatured calf thymus DNA (working solution; see recipe)
  • Dulbecco's PBS containing 5% FBS (DPBS/5% FBS)
  • Positive reference control: e.g., serum from an autoimmune strain of mice, such as MRL/lpr
  • Serum samples from mice with CGVHD
  • recipeHorseradish peroxidase–conjugated goat anti–mouse IgG (HRP‐g‐anti‐mIg; see recipe)
  • 96‐well flat‐bottomed microtiter plate
  • ELISA microtiter plate reader
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Figures

Videos

Literature Cited

Literature Cited
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