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.
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.
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 and biologics at early phases of development6. Mice are ideal research animals for genetic manipulation studies because they are genetically well defined, which is ideal for studying biological pathways and mechanisms6. Several mouse major histocompatibility complex (MHC) MHC-mismatched models of GVHD have been well established, such as C57BL/6 (H2b) to BALB/c (H2d) and FVB (H2q)→C57BL/6 (H2b)5,7. These are particularly valuable models to determine the role of individual cell types, genes, and factors that affect GVHD. Transplantation from C57/BL/6 (H2b) parental donors to recipients with mutations in MHC I (B6.C-H2bm1) and/or MHC II (B6.C-H2bm12) revealed that a mismatch in both MHC class I and class II is an important requirement for the development of acute GVHD. This suggests that both CD4+ and CD8+ T-cells are required for disease development7,8. GVHD is also involved in an inflammatory cascade known as the 'pro-inflammatory cytokine storm'9. The most common conditioning method in murine models is total body irradiation (TBI) by X-ray or 137Cs. This leads to the recipient's bone marrow ablation, thereby allowing donor stem cell engraftment and preventing rejection of the graft. This is done by limiting the proliferation of recipient T-cells in response to donor cells. Additionally, genetic disparities play an important role in disease induction, which also depends on minor MHC-mismatch10. Therefore, myeloablative irradiation dose varies in different mouse strains (e.g., BALB/c→C57BL/6).
Activation of donor T-cells by host antigen presenting cells (APCs) is essential for GVHD development. Among the APCs, dendritic cells (DCs) are the most potent. They are inheritably capable of inducing GVHD due to their superior antigen uptake, expression of T-cell co-stimulatory molecules, and production of pro-inflammatory cytokines that polarize T-cells into pathogenic subsets. Recipient DCs are critical for facilitating T-cell priming and GVHD induction after transplantation11,12. Accordingly, DCs have become interesting targets in the treatment of GVHD12.
TBI is required to enhance the donor cell engraftment. Due to the TBI effect, recipient DCs are activated and survive for a short time after the transplantation12. Despite major advancements in the usage of bioluminescence or fluorescence, establishing an effective model to study the role of recipient DCs in GVHD is still challenging.
Because donor T-cells are the driving force for GVL activity, treatment strategies using immunosuppressive drugs such as steroids to suppress T-cell alloreactivity often cause tumor relapse or infection13. Therefore, targeting recipient DCs may provide an alternative approach to treat GVHD while preserving the GVL effect and avoiding infection.
In brief, the current study provides a platform to understand how different types of signaling in recipient DCs regulates GVHD development and the GVL effect after BMT.
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 …
The authors have nothing to disclose.
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.
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 |