Optimizing graft source for allogeneic hematopoietic stem cell transplantation 公开

Hassan, Mojibade (Fall 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/zk51vh936?locale=zh


Allogeneic hematopoietic stem cell transplantation (HSCT) is curative for patients diagnosed with blood cell malignancies and genetic blood disorders. Hematopoietic stem cells (HSC) are obtained either from bone marrow (BM) aspiration or are collected by apheresis post daily administration of the mobilizing agent, granulocyte-colony stimulating factor (G-CSF). Clinical outcomes for survival and acute graft-versus-host disease (GvHD) amongst recipients of bone marrow versus G-CSF-mobilized peripheral blood (G-mobilized) grafts are similar, but higher incidence and greater severity of chronic GvHD is seen in recipients of G-mobilized grafts. A critical, unmet need in the field of HSCT is understanding how graft source selection results in the highest rates of survival without inducing disease relapse, graft rejection, or acute or chronic GvHD. Because the immune cell content of stem cell grafts has been correlated with development of GvHD, modulating immune cell content is a possible method to improve the success of stem cell transplants. The graft content of donor plasmacytoid dendritic cells (pDC) has been associated with improved transplant outcomes including decreased acute GvHD in both humans and murine models.  The manner in which pDC are thought to prevent GvHD will be discussed herein. 

Results of the clinical study, BMTCTN0201, revealed that BM grafts that contained higher than median numbers of pDC resulted in decreased incidence of chronic graft-versus-host disease (GvHD) in allogeneic hematopoietic stem cell transplant recipients. This association was not seen in recipients of G-mobilized grafts. We hypothesized that observed differences in incidence and severity of chronic GvHD amongst recipients of BM and G-mobilized grafts are due to immune-regulatory properties of pDC. Performing murine transplants with fluorescent-activated cell sorting (FACS)-purified BM or G-mobilized pDC, combined with HSC and T cells, we show that donor BM pDC limit GvHD and improve survival compared with G-mobilized pDC. BM pDC expressed higher levels of CCR9 and secreted more IL-12 than G-mobilized pDC. Using CCR9 knockout or IL-12 knockout donor mice as the source of pDC caused severe GvHD in transplant recipients and greatly reduced survival. Additionally, gene expression analysis showed BM pDC downregulated pathways regulating T cell polarization and co-stimulation compared with G-mobilized pDC. In contrast, G-mobilized pDC had greater antigen-presenting abilities and induced higher levels of inflammatory cytokines, ultimately decreasing the content of regulatory T cell in transplant recipients. In graft-versus-leukemia (GvL) models, survival, and tumor burden were similar comparing recipients of donor BM to recipients of G-mobilized pDC. Thus, BM pDC were able to limit GvHD without diminishing GvL. These data provided mechanistic insights on differences in how donor pDC regulate T cell allo-reactivity and chronic GvHD in BM versus G-mobilized allo-transplants. 

Although donor BM pDC increase survival and decrease GvHD compared to G-CSF-mobilized grafts, not all BM grafts have higher than median numbers of pDC. Thus, we developed a method to increase pDC content in BM grafts. We studied the effects of FMS-like tyrosine kinase 3 ligand (Flt3L) treatment of murine BM donors on graft pDC content and on transplant outcomes. Flt3L treatment (300μg/kg/day) caused an increase in the content of pDC in the bone marrow of donor mice in a dose dependent manner.  A treatment schedule of 300μg/kg/day on days -4 and -1 was chosen because we observed greater than 5-fold increase in pDC content without significant increase in content of hematopoietic stem cells, T cells, B cells, and natural killer cells in the bone marrow graft. Additionally, utilizing an MHC mismatched murine transplant model, recipients of Flt3L-treated BM (F-BM) and T cells had increased survival and decreased GvHD scores. Furthermore, recipients of F-BM grafts had less Th1 and Th17 polarization in donor T cells at early time-points post-transplant compared with recipients of BM and T cells. Recipients of FACS-isolated pDC from F-BM reduced GvHD and improved survival compared to recipients of equivalent numbers of untreated marrow pDC. Furthermore, upregulation of adaptive immune pathways and immunoregulatory checkpoints was observed in gene array analyses of purified F-BM pDC compared with pDC from untreated BM donors. Finally, recipients of F-BM grafts had similar tumor burden and prolonged survival in two murine GvL models. Therefore, we show that bone marrow pDC result in improved HSCT outcomes and Flt3L treatment of marrow donors is a novel method to increase the content of pDC in allografts, increase survival, and decrease GvHD without diminishing GvL effect. These insights will further the understanding of the impact of graft source on transplant outcomes and has the potential to inform clinical practice in this field.

Table of Contents

Chapter 1: Introduction                                                                                                1

1-1-0    Hematopoietic Stem Cell Transplantation                                               2

1-1-1    Overview of Hematopoietic Stem Cell Transplantation               2

1-1-2    Hematopoietic Stem Cell Transplantation Immunology               4

1-1-3    Complications of Hematopoietic Stem Cell Transplantation        6

1-1-4    Regulation of Post-Transplant Immunity                                     8

1-2-0    Hematopoietic Stem Cell Mobilizing Agents                                            10

1-2-1    Overview of Granulocyte Colony Stimulating Factor                 10

1-2-2    Overview of FMS-like Tyrosine Kinase 3 Ligand                       11

1-3-0    Plasmacytoid Dendritic Cells and Their Role in Transplant                   12

1-3-1    Overview of Plasmacytoid Dendritic Cells                                 12

1-3-2    Plasmacytoid Dendritic Cells in Immunity                                   14

1-3-3    Plasmacytoid Dendritic Cells in Transplant                                16

1-4-0    Murine Hematopoietic Stem Cell Transplant Models                              19

1-4-1    Benefits of Murine Hematopoietic Stem Cell Transplant Models 19

1-4-2    Murine Hematopoietic Stem Cell Transplant Models Limitations 21

1-5-0    Figures and Figure Legends                                                                   22

1-5-1    Figure Legends                                                                            22

1-5-2    Figures                                                                                         23

Chapter 2: Donor Bone Marrow, not Granulocyte Colony Stimulating Factor   Plasmacytoid Dendritic Cell Increase Survival and Reduce Graft-versus-Host Disease in Murine Allogeneic Hematopoietic Stem Cell Transplantation              26

           2-1-0  Abstract                                                                                                   27

           2-2-0  Introduction                                                                                              28

           2-3-0  Materials and Methods                                                                            30

           2-4-0  Results                                                                                                     35

           2-4-0  Discussion                                                                                               42

           2-5-0  Figure Legends                                                                                       46

           2-6-0  Figures                                                                                                     50

2-7-0  Supplementary Figure Legends                                                             58

2-8-0  Supplementary Figures                                                                           60

Chapter 3: FMS-like Tyrosine Kinase 3 Ligand Increases Plasmacytoid   

Dendritic Cell Content in Bone Marrow Grafts and Increases Survival and Decreases GvHD in Allogeneic Hematopoietic Stem Cell Transplantation           66

           3-1-0  Abstract                                                                                                 67

           3-2-0  Introduction                                                                                            68

           3-3-0  Materials and Methods                                                                          70

           3-4-0  Results                                                                                                   75

           3-4-0  Discussion                                                                                             80

           3-5-0  Figure Legends                                                                                     83

           3-6-0  Figures                                                                                                  86

           3-7-0  Supplementary Figure Legends                                                            92

           3-8-0  Supplementary Figures                                                                         94

3-9-0  Tables and Table Legends                                                                    99

Chapter 4: Discussion                                                                                                103

           4-1-0  Summary                                                                                               104

           4-2-0  Conclusions                                                                                           106

           4-3-0  Future Directions                                                                                   111

References                                                                                                                   115

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