The immune system protects us from invading pathogens, but has to be kept in check to prevent harmful responses to our own tissues. Unique immune “suppressor” cells have been recently characterized that prevent harmful responses to our own cells and proteins. We have recently identified unique populations of white blood cells, called dendritic cells that can induce the development of such “suppressor” cells. More importantly these immunosuppressive dendritic cells are unique in that they have special molecules on their cell surface that target them to tissues in the body where autoaggressive immune cells are either killed off or shut down during the development of the immune system.
We wish to (i) expand and isolate populations of dendritic cells based on their cell surface expression of these special trafficking molecules and then (ii) use them as facilitator cells in transplants of stem cells from a foreign donor. The idea behind our approach in stem cell transplantation therapy is that immunosuppressive dendritic cells from donor A will educate the immune system in recipient B to recognize cells and proteins from the donor as self and not to mount destructive rejection responses to stem cell grafts from the same donor (A).
Finally we wish to use a preservation protocol in formalin-based solvents to maintain the immunosuppressive attributes of these dendritic cells forever. That way they can be safely administered to patients that receive mismatched stem cell grafts without ever worrying that the function of these dendritic cells will change after injection into the patient. We will work to establish protocols for the use of immunosuppressive dendritic cells as facilitator cells for foreign stem cell grafts in the clinic.
The use of stem cell grafts has been pioneered in the clinics and biotech labs (e.g. Geron) of California and has been a therapy of choice for the replacement of diseased tissue in many patients. With the ban lifted on embryonic stem cell research, we will see more patients throughout California embracing this form of therapy. However rejection of foreign tissue grafts, including stem cells, has challenged researchers and clinicians to understand the cellular and molecular mechanisms responsible for immune-mediated rejection of foreign grafts. The preliminary results from studies described in this proposal suggest that the host immune system can be educated to recognize foreign stem cell grafts as “self” if the host is first pre-conditioned with facilitator cells obtained from the donor. The studies described in this proposal seek to identify potent facilitator cells for use in transplant therapy. This approach will allow patients throughout California that suffer from ailments that involve diseased tissue, from neurodegenerative and autoimmune disorders to immune cell cancers, to receive foreign stem cell grafts without the complications of graft rejection.
This proposal aims to overcome the challenge of selecting immature dendritic cells, which mediate tolerance, without causing the cells to activate and mature into immuno-stimulatory dendritic cells. The investigators propose to use antibodies that bind to novel markers that differ from known co-stimulatory molecules such as CD40 and may have immunomodulatory effects. Three aims are proposed: 1) to identify and validate non-activating markers for the isolation of mouse and human tolerogenic dendritic cells, 2) to compare the phenotype and tolerogenic function of two human peripheral blood dendritic cell populations identified by two respective chemokine receptor markers, and 3) to assess the utility of fixation to stabilize the tolerogenic phenotype of dendritic cells after isolation.
Reviewers noted that the concept of dendritic cell-based therapy as a way to influence the immune response is not new and that others in the field have made numerous attempts with some success in various animal models, but to date little impact has been accomplished in human studies. However, the suggestion in this proposal that trafficking receptors could be used for selection of tolerogenic dendritic cells without affecting their function was thought to be a novel and creative approach. If successful in either controlling graft versus host disease (GVHD) or preventing graft rejection under reduced conditioning, the work could greatly contribute to overcoming rejection of hematopoietic stem cells without GVHD and to the establishment of durable hematopoietic chimerism. Success of this approach is highly dependent on the presumed inactivity of the trafficking receptors and tolerance potential of fixed cells; reviewers were doubtful that this would be the case and, therefore, were not convinced that the new markers offered any advantage over past efforts to exploit dendritic cells. A reviewer noted that dendritic cells normally play a part in presenting antigens for peripheral tolerance in vivo, but the idea that dendritic cells could tolerize to many alloantigens in the transplant situation is something that has not yet been achieved without added immunosuppression, and there was little confidence that it could be achieved with this approach.
Reviewers believed that the experiments outlined were generally achievable and would provide meaningful results, but there was concern that the data would have little relevance to the human situation. One reviewer felt that the rationale for the development and testing of tolerance strategies was logical, convincing and based on strong scientific evidence and that the investigators provided convincing data in a mouse model for GVHD. However, reviewers raised several technical concerns that they felt were not adequately addressed by the applicant. First, reviewers were concerned that the antibodies to the trafficking receptors might actually affect dendritic cell activation or their proper homing after in vivo infusion, which might impact their effectiveness. Second, the process of fixation might also affect proper homing of cells, and it was unclear how the fixed cells would actually achieve tolerance. Some theories suggest that dendritic cells need to take up antigen and present it to T-cells to induce antigen-specific or allo-specific immune responses; this function would presumably be lost upon fixation. The preliminary data did not provide possible insights into mechanisms underlying tolerance induction by fixed cells and alternative plans regarding potential problems with the fixation protocol were not fully developed. Third, one reviewer indicated that testing for alloreactivity in vitro as proposed has not proven to be very indicative of in vivo responses and therefore may not be informative in these studies. For example, mixed lymphocyte reaction between allogeneic donor and recipient has not generally predicted development of GVHD or graft versus leukemia (GVL) after in vivo transplantation. Generation of tolerance in GVHD would not necessarily be the same as tolerance for other tissue types such as would be needed for tissues made from pluripotent stem cells. Fourth, the applicants suggest that HLA-matched banking of tolerogenic dendritic cells could work, but this ignores minor non-HLA antigens that are clearly the targets of GVHD and GVL. Fifth, there was no data or discussion on numbers of tolerogenic dendritic cells needed to have therapeutic benefit.
The PI is a junior investigator with reasonable experience in lymphocyte and dendritic cell trafficking, and in GVHD models of transplantation. A co-investigator brings a complementary background in tolerance and stem cell transplantation. The PI will devote 25% effort to project. The PI has a patent filed related to dendritic cells, minimal publications in total but one high impact paper and two manuscripts in preparation. A key and well-recognized co-investigator on the project will contribute 8% effort and has mentored the PI. The facilities are adequate; the PI will be working in space assigned to the co-investigator with access to necessary instruments and equipment.
Overall, reviewers felt that the proposal presented an interesting and novel approach for isolating and preparing immature dendritic cells for tolerance induction. The aims are achievable but success relies on the proposed trafficking antibodies and fixation protocols having minimal effect on the function of dendritic cells to induce tolerance in vivo. Reviewers were not fully convinced that the isolated dendritic cells would retain their desired function in vivo and were also concerned that study finding may not translate to use in humans. The PI and research team were thought to be qualified and capable of carrying out the proposed work.
A motion was made to move this application into Tier 1, Recommended for Funding. The maker of the motion argued that this proposal comes from young independent investigator who has a novel approach for using dendritic cells to induce tolerance and that this area of study is not represented among the current Tier 1 applications. Other reviewers argued that there is already a lot of ongoing work in this area by different groups and results from such work shows that tolerance only seems to work with added immunosuppression. The motion was opposed by a majority of the GWG. Because the motion was supported by more than 35% of members, supporters have exercised their right to have that position reported to the ICOC.
The minority position presents three reasons to recommend this application for funding. First, this is the only application in Tier 1 addressing the use of dendritic cells for tolerance induction; this important approach should be represented in the program even if found to have some flaws in the design. Second, by funding this application CIRM will promote the career of a bright young investigator. Third, supporting this application will encourage entry of a renowned basic immunologist (co-investigator on project), into the translational area of tolerance induction to stem cells. Based on this individual’s previous seminal contributions there is high likelihood that this important area of investigation will be enhanced.