Bone Marrow Grafts from Donors Treated with Flt3L Have More Plasmacytoid Dendritic Cells and Lead to Improved Transplant Outcomes Restricted; Files Only

Ulezko Antonova, Alina (Spring 2019)

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Hematological malignancies can be cured with hematopoietic stem cell transplantation (HSCT). The source of hematopoietic stem cells (HSC) from adult donors is either bone marrow (BM) or granulocyte-colony stimulating factor mobilized peripheral blood (G-PB). A randomized, multi-center clinical trial (BMT CTN 0201) showed better overall survival (OS) and less graft-versus-host disease (GvHD) among recipients of grafts containing higher numbers of plasmacytoid dendritic cells (pDC). This was only true for recipients of BM grafts, but not recipients of G-PB grafts. Thus, BM pDC may be more effective than G-PB pDC in regulating post-transplant GvHD. Unfortunately, not all BM grafts have optimal numbers of pDC. Therefore, a method to increase pDC numbers in all BM grafts is highly significant and may offer a novel approach to prevent chronic GvHD (cGvHD). Because FMS-like receptor tyrosine kinase 3 ligand (Flt3L) up-regulates pDC proliferation and maintains pDC homeostasis, we hypothesized that Flt3L could be used as a method to increase pDC content in BM grafts. We show that s.c. administration of 300 mg/kg of Flt3L on days -4 and -1 to mice increases >3 fold the content of pDC in BM grafts without affecting the content of HSCs. Using an allogeneic transplant model (C57BL/6àB10.BR), we report that transplantation of 5 million T-cell depleted Flt3L-stimulated BM (TCD F-BM) cells plus 4 million allogeneic donor T cells resulted in improved overall survival and decreased GvHD as compared to control TCD BM plus T cells. Donor-derived T cells in recipients of F-BM were marked by decreased Th1 and Th17 polarization. Comparative analysis of mRNA from pDC sorted from human and murine donors treated with Flt3L indicated up-regulation of immunoregulatory checkpoints and adaptive immune pathways, as well as genes involved in intrathymic thymocyte selection as compared to pDC from control untreated marrow. Moreover, Flt3L-treated donors had increased intra-thymic pDC content, and allogeneic transplantation of sorted pDC from eGFP+ mice revealed increased homing of donor-derived pDC to the recipient thymus. In conclusion, we report that donor BM graft treatment with Flt3L may improve HSCT outcomes by increasing pDC content and their GvHD-regulating activity, possibly by pDC involvement in the elimination of auto-reactive T cells that would otherwise initiate GvHD.

Table of Contents

Table of Contents

I.              Introduction

1.    Hematopoietic Stem Cell Transplantation                                         1      

1.1.        Overview of HSCT                                                                 1

1.2.        Clinical Harvesting of Cells in HSCT: BM vs G-CSF        2

1.3.        Misconceptions Around the Degree of Donor Affectation Following the Two Procedures       3

1.4.        Complications of HSCT                                                         4

1.5.        Survival and GvHD after Transplant                                  5

2.    Plasmacytoid Dendritic Cells                                                      6

2.1.        Overview of pDC                                                                    6

2.2.        Role of pDC in Transplant Tolerance                                7

3.    Pharmacological Strategies to Modify pDC Graft Content and Function 8

3.1.        FMS-Like Tyrosine Kinase 3 Ligand                                   9

3.2.        Effect of Flt3L on pDC                                                          11

4.    Potential Mechanisms for Donor pDC to Regulate Chronic GvHD 12

4.1.        Possible Role of the Thymic pDC in HSCT                      12

II.            Hypothesis                                                                                   14

III.          Materials and Methods                                                               14

1.    Mice                                                                                                        14

2.    Flt3L Treatment                                                                                    15

3.    T Cell Depletion and Purification                                                     15

4.    HSC and pDC Purification                                                                  15

5.    pDC Characterization                                                                          16

6.    Bone Marrow Transplantation                                                           17

7.    Cytospin                                                                                                 18

8.    Tumor Cell Challenge and Bioluminescent Imaging                    18

9.    Gene Array Analysis                                                                            19

10. Thymic Collection and Confocal Microscopy Analysis of Recipient Thymi    21

IV.         Results                                                                                                   21

V.           Figure legends                                                                                        35

VI.         Figures                                                                                                        41

VII.       Discussion and Future Directions                                                           51

VIII.     Works Cited                                                                                                  57

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