Suzanne T. Ildstad, M.D. Research Interests
The transplant immunology and stem cell biology program is designed to develop research that will allow for a swift transition of the discoveries made in the laboratories into a clinical setting. Experiments are focused on developing safe and effective methods for bone marrow transplantation. A considerable amount of work has been aimed at developing an improved understanding of the mechanisms that allow engraftment to occur following bone marrow transplantation using mismatched haploidentical bone marrow from which hematopoietic stem cells (HSC) and a novel population of graft facilitating cells (FC) are isolated. Studies are underway to establish the mechanism of action of murine FC and human FC. Additional studies underway include: evaluating partial conditioning of the recipient to allow "space" for graft acceptance, achieving mixed chimerism in sensitized recipients as a model to cure sickle cell disease, using bone marrow transplantation as a means to cure autoimmune diseases, such as type I diabetes, applying the establishment of mixed chimerism to induce tolerance and evaluating the role of HSC in tissue regeneration.
A combination of in vivo, in vitro and molecular techniques are currently being used to help us elucidate what role or roles the FC may be playing in allogeneic stem cell engraftment. Long-term cell cultures allow us to evaluate the effects of various conditions on HSC survival. In addition, we utilize knockout and transgenic mice - mice with specific, targeted immunological deficiencies - to explore the FC phenotype, as well as the relationship between the FC and the HSC. Our ability to sort highly purified cellular populations using flow cytometry greatly enhances our exploration of FC function and ontogeny. We have been studying the developmental pathway of the FC using GFP + (green fluorescent protein) transgenic mice, a valuable tool for tracing the FC in vivo.
Without facilitating cells, HSC do not engraft durably in mismatched recipients. We hypothesize that FC directly or indirectly regulate HSC or the recipient microenvironment to retain self-renewal and pluripotency of the HSC. Our work this year has led us to a deeper understanding of the FC as a heterogeneous population. Once we characterize the FC and fully understand the FC's phenotype, we can optimize the composition of the FC population and amplify the FC by ex vivo expansion for potential clinical application.
PARTIAL CONDITIONING TO ALLOW "SPACE" FOR GRAFT ACCEPTANCE AND DONOR-SPECIFIC TOLERANCE. H. XU
Solid organ transplantation is now routine for treatment of end-stage organ failure. Currently, the donor organ shortage, graft rejection, and deleterious side effects associated with the use of nonspecific immunosuppression remain the major limitations in transplantation. The induction of donor-specific tolerance with hematopoietic chimerism may offer a potential solution for these limitations. The clinical application of this technique is limited by the morbidity and mortality of conventional bone marrow transplantation (BMT). The conditioning for engraftment of hematopoietic stem cells is nonspecific, utilizing myeloablation plus nonspecific immunosuppression. Recently, non-lethal or partial conditioning strategies have been demonstrated to induce mixed chimerism and donor-specific tolerance in a number of animal models. If engraftment could be achieved with less toxic strategies, the application of chimerism to induce tolerance would be more suitable for clinical use.
The purposes of the experiments performed in this project are to characterize which cells in the recipient hematopoietic microenvironment prevent marrow engraftment. These data may provide new strategies to further reduce the requirements of conditioning. Further, studies are underway that may allow the engraftment to occur by targeting these defined specific effector cells with monoclonal antibodies (mAb). We were the first to propose a partial conditioning approach to intentionally establish mixed chimerism in mice. Since that report nearly 12 years ago, others have confirmed our work in dogs, pigs, and now humans.
OPTIMIZED FACILITATING CELL/STEM CELLS TO INDUCE TOLERANCE FOR TRANSPLANTATION. I. FUGIER-VIVIER, F. REZZOUG, Y. HUANG, C. SCHANIE, AND MARC DY-LIACCO.
Islet transplantation is the preferred approach to maintain glucose homeostasis in patients with type 1 diabetes. However, rejection of the transplanted tissue has posed a major limitation to widespread application of the approach. Mixed chimerism induces tolerance to solid organ and cellular transplants. However, graft-versus-host disease (GVHD) has limited the clinical application of chimerism to induce tolerance. The discovery of facilitating cells (FC) is an important finding for the clinical application of chimerism since engraftment can be enhanced, GVHD avoided, and safe transplants allowed in mismatched recipients. Although potent in biologic effect, the CD8 + /TCR - FC population is heterogeneous. The goal of this research is to more precisely identify the cell subtype(s) in the CD8 + /TCR - facilitating cell population in order to make the clinical application of allogeneic BMT to induce tolerance safer and widely available. The findings from these studies will lead to therapeutic strategies to produce a more potent tolerance-inducing clinical regimen with fewer side effects for islet transplantation and other organ grafts.
SICKLE CELL DISEASE AND THE SENSITIZED STATE. H. XU
Sickle cell disease (SCD) is the most frequently inherited hemoglobinopathy. The clinical course is chronic andle cell disease die by age 40. Mixed chimerism induced by bone marrow debilitating, and requires frequent hospitalizations. In the United States , 50% of people living with sick transplantation (BMT) cures SCD. With mixed chimerism, it may be possible to expand the donor pool and avoid the morbidity associated with full ablation. One limitation to BMT for SCD is that the recipients are often highly sensitized to HLA alloantigens due to chronic transfusion therapy. Sensitized recipients are more prone to bone marrow as well as solid organ graft rejection. Currently, patients with SCD who have developed renal failure are not considered to be candidates for renal transplantation or BMT because of their disease. Mixed chimerism may reverse the immunologic memory and establish donor tolerance.
The aims of this project are to develop a clinically relevant approach to achieve mixed hematopoietic stem cell chimerism in highly sensitized recipients and to evaluate whether pre-sensitized recipients who receive a BMT are tolerant to their donor. We have established the sensitized animal model by pre-skin grafting. Our goal is to achieve mixed allogeneic marrow engraftment and donor-specific tolerance in these pre-sensitized animals with partial conditioning strategies. Studies are currently underway to test certain monoclonal antibodies in combination with immunosuppressive drug treatment which will allow for the establishment of mixed chimerism in presensitized recipients.
IDENTIFICATION OF HUMAN FACILITATION CELLS. R. REZZOUG, I. FUGIER-VIVIER, M. TANNER, IN COLLABORATION WITH C. KAUFMAN, M.J. ELLIOTT, W. DARAG, AND R. HERZIG
The goal of this project is to identify the facilitating cell population in human bone marrow (HuFC). As described above, mouse bone marrow FC have been identified and characterized, and can be mobilized into the peripheral blood following growth factor treatment (Flt3 ligand, G-CSF).
We currently have IRB approved protocols to receive vertebral columns from deceased organ donors, as well as "waste" mobilized peripheral blood, for research purposes.
The FC function in mice to facilitate hematopoietic stem cell (HSC) engraftment in allogeneic recipients, which we study in our in vivo murine model. Since we do not have a corresponding in vivo assay in the human, we have to develop an in vitro assay using mice that can be used with the HuFC. We are currently evaluating HuFC from both the bone marrow and mobilized peripheral blood for the phenotype, the cell composition and the in vitro function. HuFC are found in the lymphoid population of BM and are isolated by CD8 + , CD56 - /dim and TCR - surface expression.
Future avenues of investigation will be to evaluate HuFC function in a xenogeneic model (human to mouse) using SCID mice as recipients. The identification of the HuFC and the determination of their role in HSC engraftment would be of paramount interest in the field of bone marrow and stem cell transplantation.
DIABETES AND AUTOIMMUNITY. Y. HUANG, F. REZZOUG, C. SCHANIE, AND I. FUGIER-VIVIER (IN COLLABORATION WITH DR. MARIUSZ RATAJCZAK'S TEAM)
Diabetes affects approximately 16 million Americans. Diabetes is caused by the lack of available insulin and thus the inability to maintain healthy blood sugar levels. Patients with type I diabetes (also known as juvenile or insulin-dependent diabetes) lose their ability to produce insulin. This is caused by the destruction of the insulin-producing cells (ß cells) in the pancreas. The ß cells are targets of an autoimmune attack. During this attack the diabetic's own immune cells, especially the T cells, recognize unique proteins in the ß cells as foreign and go about ridding the ß cells from the pancreas, as they would a cell infected with a bacteria or virus.
Type I diabetes and other autoimmune diseases are caused by defects in the primitive cells that are the precursor cells of the blood and immune system called hematopoietic stem cells. Hematopoietic stem cells are capable of producing the entire set of cells that comprises the immune system, as well as red blood cells and platelets. Since defects in the hematopoietic stem cell cause many autoimmune diseases, these diseases can actually be CURED through bone marrow transplantation (BMT). Both experimental as well as clinical studies have shown that BMT can eliminate the autoimmunity that causes several different autoimmune diseases, including type I diabetes, systemic lupus erythematosis (lupus or SLE), rheumatoid arthritis, Crohn's colitis, and an animal disease model similar to multiple sclerosis.
Previous studies by our laboratory have shown that BMT halts the autoimmune processes and reverses the damage caused by autoimmune cells in nonobese diabetic (NOD) mice. These studies also showed that complete replacement of the NOD immune cell system is not required to prevent diabetes development. BMT recipients that possess cells of both donor- and recipient-origin (mixed chimeras) resolve their autoimmune disease as well as those that only exhibit cells of donor-origin. Additionally, these previous studies have shed light on various deficiencies exhibited by the bone marrow of NOD.
Bone marrow from nonobese diabetic (NOD) mice lacks a specific subset of cells that mature into antigen-presenting cells. This subset can be defined by the cell surface expression of heat stable antigen (HSA) and Ly6C. Transplantation of NOD mice with bone marrow from non-diabetic strains of mice results in the cure of autoimmunity and diabetes. When the bone marrow from recipient NOD mice was tested for the presence of the HSA/Ly6C populations after transplantation, the population was restored. NOD bone marrow cells cultured with the cytokine Flt3-ligand also saw a restoration of the HSA/Ly6C population. Interestingly, In vivo treatment with Flt3-ligand restores the HAS/Ly6C population in the mice and delays the development of diabetes. These data, taken together, suggest that the underlying myeloid abnormality in NOD mice is due to a block in development of myeloid progenitor cells, which is restored through treatment with FL and may provide a benign and novel approach to treat autoimmune diabetes.
Mixed chimerism induces tolerance to islet transplants. Our goal, in collaboration with Dr. Ratajczak, is to develop a novel approach to establish chimerism in NOD mice by inducing immune deviation to promote host-versus-graft hyporesponsiveness with minimal conditioning, thereby giving the HSC an opportunity to engraft and establish subsequent self-perpetuating deletional tolerance. Additionally, a great deal of recent interest has been focused on the therapeutic potential of cell-based therapies to induce tolerance. We are exploring ways to use pre-transplant immunomodulation of the donor with hematopoietic growth factors to promote tolerance.
Current work in the laboratory has three distinct goals: (1) to decrease the risks of BMT for the treatment of autoimmune diseases using the NOD mouse model; (2) to characterize the mechanisms underlying the deficiencies described in NOD mouse bone marrow; and (3) to induce tolerance to islet transplants.
GENETICS OF HSC ENGRAFTMENT (FURTHER CHARACTERIZATION OF FC. Y. HUANG AND F. REZZOUG
The major complications of purified HSC transplantation include graft rejection and graft failure. Engraftment of highly purified HSC in MHC-matched recipients is different from that for MHC- disparate allogeneic recipients. Previous studies have demonstrated that facilitating cells (FC) (CD8+/TCR-) enhance engraftment of purified HSC in allogeneic recipients without causing GVHD. It is hypothesized that a specific MHC molecule strongly influences engraftment of HSC mediated by FC. In the present studies, we evaluated which MHC loci are essential for durable engraftment of purified allogeneic HSC.
The MHC in the mouse is defined by the K and D loci of class I and I-A and I-E loci of class II. Donor and recipient strains were chosen based on matching at selected MHC loci. Our data indicate that matching at class I D is not essential for HSC engraftment. If the donor and recipient are disparate at the class I K and class II-A locus, impaired engraftment of HSC results. The matching for class I K appears to be the most critical, as reflected in failure of long-term graft survival in B10.BR -> B10.MBR (class I K disparate). These data demonstrate that the MHC class I K is the critical molecule for engraftment of purified allogeneic HSC. We then examined which MHC loci are important to FC: HSC interaction and/or function and whether matching between FC and HSC would allow durable HSC chimerism. Our data also demonstrate that MHC class I K is an important molecule for FC and HSC interaction. When the FC and HSC are matched at the class I K locus, matching in the other loci is not required, as evidenced by durable engraftment in allogeneic recipients. The fact that FC enable engraftment of HSC when matched at class I K supports a trophic mechanism of action over a veto mechanism.
EX-VIVO EXPANSION USING FLT3-LIGAND. Y. HUANG
The purpose of this project is to expand facilitating cells (FC). Flt3 ligand (FL) is a growth factor for hematopoietic progenitor and stem cell (HSC) mobilization in vivo. We previously reported that treatment of mice with FL alone and FL + G-CSF induces significant expansion of FC and HSC in the marrow compartment and peripheral blood progenitor cells (PBPC). Both growth factors showed a highly significant synergy on the mobilization of FC and HSC. The kinetics for mobilization was similar for FC and HSC, with a peak occurring on day 10. The engraftment-potential of peripheral blood mononuclear cells (PBMC) mobilized with FL and FL + G-CSF was superior to PBPC obtained from animals treated with G-CSF. We have recently evaluated the function of FL-expanded FC harvested from both compartments. FC were sorted from donors after 10 days of mobilization. FL treatment of the donor B10.BR mice resulted in a 5.8-fold increase of FC in marrow compared to normal mice. FC were also present in peripheral blood by a 15-fold increase. When FC were collected from the peripheral blood of FL-treated donors and co-administered with normal HSC, 87% of the recipients exhibited excellent engraftment and long-term survival. In contrast only 14% of the animals transplanted with normal HSC plus FL-treated FC from the marrow survived greater than three months. These results indicate that FL can expand FC in both marrow and peripheral blood. However, the FC contained in the peripheral blood after FL-treatment are functional facilitators, while those from the marrow have lost their function. Studies this year have been designed to identify the mechanism responsible for the change in biologic activity of FL-expanded FC in the marrow compartment as compared to the periphery.
COMPOSITE TISSUE TRANSPLANTATION. W.C. HUANG (IN COLLABORATION WITH THE HAND TRANSPLANT TEAM: W. BREIDENBACH, D. PIDWELL, AND G. TOBIN)
Each year more that seven million individuals undergo complex reconstructive operations to repair large composite tissue defects from trauma, surgical excision of tumors, war injuries, major burns, and congenital birth defects. Often, sufficient autologous tissue is not available. Reconstruction using allografts could provide a major advantage. The Ildstad group has teamed up with the hand transplant group to explore the use of mixed chimerism to induce tolerance to hind limb transplants in rats with quite astounding success.
In the allogeneic rat-to-rat model, the depletion of a b and g d TCR + T cells from donor marrow retains engraftment-potential yet avoids graft-versus-host disease (GVHD). Using this model, we can generate chimeric rats which then exhibit tolerance to a donor-specific hind-limb allograft. To reduce the morbidity associated with lethal ablation, we are exploring strategies to reduce the conditioning regimen, including the use of monoclonal antibody treatment to remove targeted, alloreactive recipient cell types. The ultimate goal is to eliminate the use of irradiation completely as a preconditioning strategy in the recipient patient.
THE FUTURE OF STEM CELLS: TISSUE REPAIR. C. SCHANIE AND Y. HUANG IN COLLABORATION WITH THE DEPARTMENT OF OPHTHAMOLOGY AND VISUAL SCIENCES (H. KAPLAN AND V. ENZMANN) AND THE DEPARTMENT OF CARDIOLOGY (R. BOLLI, B. DAWN AND Y. GUO)
Recent reports suggest that HSC may be capable of transdifferentiating into non-hematopoietic cell types in response to tissue injury. Using GFP + mice, we are able to purify HSC and then trace the migration and development of these cells in models of tissue damage.
The Department of Ophthalmology has a murine model for Adult Macular Degeneration (AMD) using sodium iodate to destroy the retinal pigmented epithelial cells of the eye. Similarly, the Department of Cardiology uses a murine model to induce cardiac infarct, resulting in ischemia and damage to cardiac muscle.
Preliminary data have shown in both models that purified HSC can home to and possibly transdifferentiate into tissue-specific cells and repair these sites of damage. Now we are currently working to understand how this repair occurs and whether the presence of FC will increase the HSC homing and the repair of the damaged sites.
Our laboratory is currently participating in a variety of collaborations with other University of Louisville faculty and laboratories. One such collaboration is with Dr. Roberto Bolli and the Department of Cardiology. We are working together to explore the possibility that highly purified GFP+ hematopoietic stem cells might be able to differentiate into other, non-lymphohematopoietic tissues.