Murine Hematopoietic Stem Cell Transplantation Models for Graft-versus-tumor, Graft-versus-host disease, and Post-transplant Immune Reconstitution
Allogeneic hematopoietic stem cell transplantation (HSCT) is an important therapy for a variety of malignancies, including leukemias, lymphomas, and advanced solid tumors, such as renal cell carcinoma, as well as a number of nonmalignant diseases, such as aplastic anemia and severe combined immunodeficiency. My laboratory uses murine HSCT models to study clinically important problems in HSCT and to test novel therapeutic strategies.
The therapeutic benefits of HSCT are not only derived from the opportunity to give higher doses of chemotherapy and/or radiation but also from a so-called graft-versus-tumor (GVT) effect, whereby the graft attacks host malignancy.
Yet HSCT is not without complications: graft-versus-host-disease (GVHD) and immune deficiencies after HSCT are important complications of allogeneic HSCT. GVHD is a progressive systemic illness with immunosuppression, wasting, and specific damage to skin, liver, intestines, and the immune system. Even with prophylaxis, most adults have some degree of GVHD after allogeneic bone marrow transplantation.
1. Strategies to Enhance Immune Reconstitution after Allogeneic HSCT
Our second area of interest involves the development of strategies to enhance immune recovery after HSCT and to prevent post-transplant infections. Transplant recipients are often profoundly immunodeficient after transplant, leaving them susceptible to a variety of bacterial, viral, and fungal infections. Consequently, improving the kinetics and quality of immune reconstitution after HSCT is of great interest.
Our data in mice demonstrate that the administration of the "lymphoid growth factor" Interleukin-7 (IL-7) post-transplant can result in a remarkable increase in T and B lymphocytes without aggravating GVHD. We are currently further analyzing the optimal dosing and timing of IL-7 administration and its effects on T and B cell recovery, GVHD, and GVT. At the same time, we are designing and conducting Phase I and II clinical trials to assess the clinical safety and therapeutical benefits of this cytokine.
In addition, we are performing studies to analyze the role of the thymus and peripheral apoptosis in immune reconstitution, and testing other cytokines and growth factors with potential immunostimulatory effects, such as keratinocyte growth factor (KGF), Interleukin 15 (IL-15), and insulin-like growth factor 1 (IGF-1).
We have also modified a technique to generate ex vivo large numbers of T cell precursors, which upon transfer will migrate to the thymus and develop into mature T cells. Finally, we are studying the role of sex steroids and the utility of chemical and physical castration in enhancing immune reconstitution. It is hoped that these preclinical studies will establish the above factors and treatments as safe and effective, and pave the way for clinical Phase I/II studies.
2. Adoptive immunotherapy with lymphoid precursor cells
Culture systems utilizing Notch1 signaling can be used for the in vitro development of T lineage cells at various differentiation stages. The most widely used of those systems is the OP9-DL1 system, which uses a mouse bone marrow stromal cell line transduced to express the Notch1 ligand Delta-like 1 (DL1) to coculture hematopoietic stem cells in the presence of IL-7 and FLT3-ligand. This system can be modified for the generation of large numbers of lymphoid progenitors committed to the T lineage for adoptive immunotherapy. We recently demonstrated that co-transplanted allogeneic OP9-DL1 derived early T progenitors can mature in an immunosuppressed host, mediating immunity including anti-tumor activity without causing GVHD [reference], and can even be transferred in the absence of allogeneic stem cells to any immunosuppressed individual irrespective of MHC disparities for adoptive 'off-the-shelf' immunotherapy. Such cells also protect the thymus from atrophy which otherwise hinders future lymphopoiesis, and can be genetically modified in vitro for targeted immunotherapy. Notch-based culture systems show promise for clinically applicable therapeutic use: they can be fully humanized and human cord blood and human adult BM derived CD34+ progenitor cells have been cultured in these systems to generate human T cells.
Culture and Transfer Process
3. Effector Cell Homing
It is widely understood that select organs, such as the liver, GI tract, and skin, are major targets of GVHD, while other organs, such as the heart, are relatively unaffected. The differential ability of relevant donor immune effector cell populations (chiefly T cells) to utilize trafficing molecules to enter various target organs is believed to play a significant role in determining this organ specificity in GVHD.
In ongoing studies, we are analyzing the role of organ-specific dendritic cells in determining to which organ alloreactive T cells will migrate. Furthermore, we are studying the physiology of effector cell homing in GVHD, including role of homing-related chemokines, such as CCR2, CCR9, and adhesion molecules, such as P-selectin and its ligand P-selectin glycoprotein ligand 1 (PSGL-1), in the mechanistic steps of homing (e.g., tethering and rolling or activation by chemokines).
Our results demonstrate that selective ablation of either chemokine receptors or selectins can significantly reduce GVHD target organ pathology and overall morbidity and mortality, while preserving GVT activity.
4. Tumor Vaccination
Although allogeneic bone marrow transplantation is often used with great therapeutic success as a treatment for a variety of liquid and solid malignancies, disease relapse remains a significant problem and continues to reduce the overall outcome in recipients of allogeneic HSCT.
In continuing collaboration with Dr. Alan Houghton's tumor immunology laboratory here at MSKCC, we are studying the efficacy of post-transplant tumor vaccination in murine HSCT recipients with the goal of enhancing the graft-versus-tumor (GVT) effect.
Although the malignant relapse rate (often indicative of the strength of the GVT effect) is usually inversely related to the severity of GVHD in a given patient, we hypothesize that immunization against specific-tumor antigens, with suitable adjuvant strategies in the post-transplant setting, may decrease the risk of malignant relapse without enhancing the risk of GVHD.
We are employing a variety of tumor vaccines, including whole-cell vaccines, peptide vaccines, and DNA vaccines. We are also studying combinations of vaccines and immune adjuvants (IL-15, anti-CTLA-4 antibody) as well as immune reconstituting agents (IL-7, KGF, T cell precursors). Our vaccine targets include melanoma antigens as well as the leukemia antigen WT1. In addition, we are modeling adoptive transfer cellular therapies in the post-transplant setting.
5. Role of Th17 Cells in Graft-versus-Host Disease
Recently a new subset of T helper cell was characterized; these cells have been identified as IL-17 producing CD4+ T cells (Th17 cells). Our lab is investigating the role Th17 cells in the development of GVHD. Identification of both specific T cell subset and the cytokine responsible for promoting GVHD may lead to potential therapies.
Th17 cells produce high levels of IL-17, IL-21 and IL-22 and require IL-23 for survival and proliferation. IL-17 is a potent proinflammatory cytokine that induces the production of other inflammatory cytokines (IL-6 and TNF-α), chemokines (KC, MCP-1 and MIP-2) and matrix metalloproteases. Additionally, IL-17 has also been shown to costimulate T cells and enhance DC maturation. The effector function of these cells includes mediating antimicrobial defense and inflammation. Studies in mice suggest that Th17 cells contribute to various automimmune diseases including rheumatoid arthritis, multiple sclerosis, lupus, asthma, inflammatory bowel disease, and colitis. Furthermore, IL-17 has been shown to play a role in allograft rejection of solid organs, thus demonstrating the proinflammatory role of Th17 cells. However, the role of Th17 cells in GVHD has not been determined.
We are currently investigating the role of Th17 cells in the development of GVHD, including IL-17 and the Th17 survival factor IL-23, which has been shown to contribute to several intestinal inflammatory diseases. Furthermore, we would like to investigate the possible clinical applications by determining the effects of IL-17 and IL-23 neutralizing antibodies in our murine model of GVHD. These studies may enlighten our understanding of intestinal GVHD.
6. Differential role of tumor-necrosis-factor (TNF) family members in GVHD and GVT
Graft-versus-host-disease (GVHD) and graft-versus-tumor effects (GVT) are both determined by the cytotoxic T lymphocytes of the donor graft. Our main goal is to find pathways, which separate the undesirable GVHD from the desirable anti-tumor functions in the post-transplantation period.
Tumor necrosis factor (TNF) is a type I cytokine involved in many pathophysiological processes; including inflammation and host defense against various pathogens. TNF can be expressed as a transmembrane molecule (memTNF) on the surface of activated and, when cleaved by the TNF- converting enzyme (TACE), is secreted by the cell in its soluble form (solTNF).
We previously found an important role of donor T cells derived TNF in the pathogenesis of GVHD. Recently, we were able to extend these findings and showed that soluble TNF (solTNF) contributes to acute GVHD, whereas membrane bound TNF (memTNF) is required for optimal graft-versus-tumor (GVT) activity. These data suggest that inhibition of secretion of solTNF might be an attractive strategy to decrease the incidence of GVHD - or even to treat established GVHD - after HSCT.
Recently, a novel class of biologics were developed (DN-TNFs) that work through a selective pharmacologic inhibition of solTNF. These drugs were found to reduce the release of inflammatory cytokines while maintaining proper immune responses against infectious pathogens. This suggests that DN-TNFs could be used to prevent GVHD without interfering with GVT or immunity against infectious pathogens. We are currently investigating DN-TNF (XNEP1595) in mouse models for GVHD, GVT and infectious diseases.
Another interesting strategy to separate GVT activity from GVHD is the enhancement of cytolytic activity on donor T cells. We were able to show that the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway was involved in GVT activity, but not GVHD, and we are currently designing strategies to enhance TRAIL activity in donor T cells to optimize their GVT potential.
We are currently optimizing a TRAIL lentivirus. This will allow us to manipulate donor T cells, resulting in increased TRAIL expression. Overexpression of TRAIL on donor T cells may enhance GVT effect after BMT.
