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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

Abstract

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient's survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Introduction

Allogeneic hematopoietic stem cell transplantation (BMT) is an effective therapy to treat hematological malignancies1,2 through the graft-versus-leukemia (GVL) effect3. However, donor lymphocytes always mount unwanted immunological attacks against recipient tissues, a process called graft-versus-host disease (GVHD)4.

Murine models of GVHD are an effective tool to study the biology of GVHD and the GVL response5. Mice are a cost-effective research animal model. They are small and efficiently dosed with molecules a....

Protocol

The experimental procedures were approved by the Institutional Animal Care and Use Committee of University of Central Florida.

1. GVHD Induction

NOTE: Allogeneic bone marrow (BM) cell transplantation (step 1.2) is performed within 24 h after irradiation. All procedures described below are performed in a sterile environment. Perform the procedure in a tissue culture hood and use filtered reagents.

  1. Day 0: Prepare the recipient mice.
    1. Use female .......

Representative Results

The major MHC-mismatched B6 (H2kb)-BALB/C (H2kd) model closely corresponded to GVHD development after the transplantation (Figure 2). All six GVHD clinical signs established previously by Cooke et al.16 occurred in the recipients transplanted with WT-B6 T-cells but not in the recipients transplanted with BM alone (step 1.5), which represented the GVHD-negative group. There are two phases in GVHD development in thi.......

Discussion

The use of stem cells to suit a particular individual is an effective approach to treat advanced and resistant cancers18. Small molecule pharmaceuticals, however, have long remained a primary focus of personalized cancer therapy. On the other hand, in cellular therapy a multitude of interactions between donor and host can decisively influence the treatment outcomes, such as the development of GVHD after BMT1.

Major MHC-mismatched mouse models of .......

Acknowledgements

This study is supported by University of Central Florida College of Medicine start-up grant (to HN), the University of Pittsburgh Medical Center Hillman Cancer Center start-up grant (to HL), the United States NIH Grant #1P20CA210300-01 and Vietnamese Ministry of Health Grant #4694/QD-BYT (to PTH). We thank Dr. Xue-zhong Yu at Medical University of South Carolina for providing materials for the study.

....

Materials

NameCompanyCatalog NumberComments
0.5 M EDTA pH 8.0 100MLFisher ScientificBP2482100MACS buffer
10X PBSFisher ScientificBP3994MACS buffer
A20 B-cell lymphomaUniversity of Central FloridaIn houseGVL experiment
ACC1 fl/flJackson Lab30954GVL experiment
ACC1 fl/fl CD4creUniversity of Central FloridaGVL experiment
Anti-Biotin MicroBeadsMiltenyi Biotec130-090-485T-cell enrichment
Anti-Human/Mouse CD45R (B220)Thermo Fisher Scientific13-0452-85T-cell enrichment
Anti-mouse B220 FITCThermo Fisher Scientific10452-85Flow cytometry analysis
Anti-mouse CD11c- AF700Thermo Fisher Scientific117319Flow cytometry analysis
Anti-Mouse CD25 PEThermo Fisher Scientific12-0251-82Flow staining
Anti-Mouse CD4 BiotinThermo Fisher Scientific13-0041-86T-cell enrichment
Anti-Mouse CD4 eFluor® 450 (Pacific Blue® replacement)Thermo Fisher Scientific48-0042-82Flow staining
Anti-mouse CD45.1 PEThermo Fisher Scientific12-0900-83Flow cytometry analysis
Anti-Mouse CD8a APCThermo Fisher Scientific17-0081-83Flow cytometry analysis
Anti-mouse H-2Kb PerCP-Fluor 710Thermo Fisher Scientific46-5958-82Flow cytometry analysis
Anti-mouse MHC Class II-antibody APCThermo Fisher Scientific17-5320-82Flow cytometry analysis
Anti-Mouse TER-119 BiotinThermo Fisher Scientific13-5921-85T-cell enrichment
Anti-Thy1.2Bio ExcelBE0066BM generation
B6 fB-/- miceUniversity of Central FloridaIn houseRecipients
B6.Ly5.1 (CD45.1+) miceCharles River564Donors
BALB/c miceCharles River028Transplant recipients
C57BL/6 miceCharles River027Donors/Recipients
CD11bThermo Fisher Scientific13-0112-85T-cell enrichment
CD25-biotinThermo Fisher Scientific13-0251-82T-cell enrichment
CD45RThermo Fisher Scientific13-0452-82T-cell enrichment
CD49b Monoclonal Antibody (DX5)-biotinThermo Fisher Scientific13-5971-82T-cell enrichment
Cell strainer 40 uMThermo Fisher Scientific22363547Cell preparation
Cell strainer 70 uMThermo Fisher Scientific22363548Cell preparation
D-LuciferinGoldbioLUCK-1GLive animal imaging
Fetal Bovine Serum (FBS)Atlanta Bilogicals R&D systemD17051Cell Culture
Flow cytometry tubesFisher Scientific352008Flow cytometry analysis
FVB/NCrlCharles River207Donors
Lipopolysacharide (LPS)Millipore SigmaL4391-1MGDC mature
LS columnMitenyi Biotec130-042-401Cell preparation
MidiMACSMiltenyi Biotec130-042-302T-cell enrichment
New Brunswick Galaxy 170R incubatorEppendorfGalaxy 170 RCell Culture
Penicilin+streptomycinPenicillin/Streptomycin (10,000 units penicillin / 10,000 mg/ml strep)GIBCO15140Media
RPMI 1640Thermo Fisher Scienctific11875-093Media
TER119Thermo Fisher Scientific13-5921-82T-cell enrichment
Xenogen IVIS-200Perkin ElmerXenogen IVIS-200Live animal imaging
X-RAD 320 Biological IrradiatorPrecision X-RAYX-RAD 320Total Body Irradiation

References

  1. Shlomchik, W. D. Graft-versus-host disease. Nature Reviews Immunology. 7, 340-352 (2007).
  2. Appelbaum, F. R. Haematopoietic cell transplantation as immunotherapy. Nature. 411, 385-389 (2001).
  3. Blazar, B. R., Murphy, W. J.,....

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