Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

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 malignancies^1,2 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 .......

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 .......

Materials

Name	Company	Catalog Number	Comments
0.5 M EDTA pH 8.0 100ML	Fisher Scientific	BP2482100	MACS buffer
10X PBS	Fisher Scientific	BP3994	MACS buffer
A20 B-cell lymphoma	University of Central Florida	In house	GVL experiment
ACC1 fl/fl	Jackson Lab	30954	GVL experiment
ACC1 fl/fl CD4cre	University of Central Florida		GVL experiment
Anti-Biotin MicroBeads	Miltenyi Biotec	130-090-485	T-cell enrichment
Anti-Human/Mouse CD45R (B220)	Thermo Fisher Scientific	13-0452-85	T-cell enrichment
Anti-mouse B220 FITC	Thermo Fisher Scientific	10452-85	Flow cytometry analysis
Anti-mouse CD11c- AF700	Thermo Fisher Scientific	117319	Flow cytometry analysis
Anti-Mouse CD25 PE	Thermo Fisher Scientific	12-0251-82	Flow staining
Anti-Mouse CD4 Biotin	Thermo Fisher Scientific	13-0041-86	T-cell enrichment
Anti-Mouse CD4 eFluor® 450 (Pacific Blue® replacement)	Thermo Fisher Scientific	48-0042-82	Flow staining
Anti-mouse CD45.1 PE	Thermo Fisher Scientific	12-0900-83	Flow cytometry analysis
Anti-Mouse CD8a APC	Thermo Fisher Scientific	17-0081-83	Flow cytometry analysis
Anti-mouse H-2Kb PerCP-Fluor 710	Thermo Fisher Scientific	46-5958-82	Flow cytometry analysis
Anti-mouse MHC Class II-antibody APC	Thermo Fisher Scientific	17-5320-82	Flow cytometry analysis
Anti-Mouse TER-119 Biotin	Thermo Fisher Scientific	13-5921-85	T-cell enrichment
Anti-Thy1.2	Bio Excel	BE0066	BM generation
B6 fB-/- mice	University of Central Florida	In house	Recipients
B6.Ly5.1 (CD45.1+) mice	Charles River	564	Donors
BALB/c mice	Charles River	028	Transplant recipients
C57BL/6 mice	Charles River	027	Donors/Recipients
CD11b	Thermo Fisher Scientific	13-0112-85	T-cell enrichment
CD25-biotin	Thermo Fisher Scientific	13-0251-82	T-cell enrichment
CD45R	Thermo Fisher Scientific	13-0452-82	T-cell enrichment
CD49b Monoclonal Antibody (DX5)-biotin	Thermo Fisher Scientific	13-5971-82	T-cell enrichment
Cell strainer 40 uM	Thermo Fisher Scientific	22363547	Cell preparation
Cell strainer 70 uM	Thermo Fisher Scientific	22363548	Cell preparation
D-Luciferin	Goldbio	LUCK-1G	Live animal imaging
Fetal Bovine Serum (FBS)	Atlanta Bilogicals R&D system	D17051	Cell Culture
Flow cytometry tubes	Fisher Scientific	352008	Flow cytometry analysis
FVB/NCrl	Charles River	207	Donors
Lipopolysacharide (LPS)	Millipore Sigma	L4391-1MG	DC mature
LS column	Mitenyi Biotec	130-042-401	Cell preparation
MidiMACS	Miltenyi Biotec	130-042-302	T-cell enrichment
New Brunswick Galaxy 170R incubator	Eppendorf	Galaxy 170 R	Cell Culture
Penicilin+streptomycinPenicillin/Streptomycin (10,000 units penicillin / 10,000 mg/ml strep)	GIBCO	15140	Media
RPMI 1640	Thermo Fisher Scienctific	11875-093	Media
TER119	Thermo Fisher Scientific	13-5921-82	T-cell enrichment
Xenogen IVIS-200	Perkin Elmer	Xenogen IVIS-200	Live animal imaging
X-RAD 320 Biological Irradiator	Precision X-RAY	X-RAD 320	Total Body Irradiation