Role of Multipotent Adult Progenitor Cells and Myeloid-Derived Suppressor Cells in regulating graft-versus-host reactivity after allogeneic haematopoietic stem cell transplantation

Allogeneic haematopoietic stem cell transplantation is a cornerstone in the treatment of various haematological malignancies. Its concept has evolved from a rescue therapy after high dose conditioning regimens to a highly effective form of immunotherapy , the success of which is based on the immune-mediated graft-versus-leukemia (GvL) response. This concept of GvL has led to the development of reduced-intensity stem cell transplantation protocols that are often combined with Donor Lymphocyte Infusions (DLI) , a form of adoptive cell-therapy given to enhance GvL in allogeneic HSCT-patients, either therapeutically or prophylactically. A GvL response is produced when donor T-cells target recipient antigen-presenting cells within the lymphohaematopoietic system, a process designated as lymphohaematopoietic graft-versus-host reactivity (LH-GvHR). A significant problem of DLI, however, is its close association with Graft-versus-Host disease (GvHD) which occurs when LH-GvHR extends to healthy epithelial tissue or bone marrow.Intensive clinical and experimental research focuses on developing alternative strategies that more specifically, safely and effectively control GvHD than the immunosuppressive drugs that are currently used. With this respect, the idea of using immunoregulatory cells that can control GvHD has emerged. The overall goal of this thesis work was to address the potential role of two novel immunoregulatory cell types, multipotent adult progenitor cells (MAPC) and myeloid-derived suppressor cells (MDSC), in the control of post-transplant GvHD. The first specific aim related to the potential use of MAPC as an off-the shelf cell product for post-transplant adoptive cell-therapy to control GvHD. The second specific aim related to the potential role of MDSC that endogenously expand in the stem cell recipient or in the stem cell donor, in the control of post-transplant GvH reactivity. In the first part of this work, we addressed the immunogenicity and immunomodulatory behavior of mouse-derived MAPC. MAPC are a specific type of adult stem cells first described by the C Verfaillie group in mice and in rats, and subsequently also in humans. MAPC are isolated from the mesenchymal stem cell population using specific culture methods. Similar to classical mesenchymal stem cells (MSC), of which the clinical success in controlling GvHD has been reported to be variable, MAPC have been proposed as an alternative cell source for adoptive immunotherapy for GvHD. MAPC exhibit an extensive proliferation potential without showing signs of senescence, which may constitute an advantage for their use as an off-the-shelf cell source in clinical applications. At the moment this work was started, few experimental data were available on the immunogenicity and immunomodulatory potential of MAPC. During the course of the current study, it was published that large-scale expanded rat MAPC (rat MultiStem®) exhibit GvHD-suppressive potential, and a large-scale expanded human MAPC (Multistem®) product has been generated. In the mean time, phase I clinical trials using Multistem® to prevent GvHD have been initiated (Clinical Trials.gov NCT00677859). In mice, two types of MAPC clones are described: Oct4pos and Oct4neg MAPC. Oct-4 is a transcription factor that is essential for the maintenance of the pluripotent state, and hence, Oct4pos MAPC have a broad differentiation profile toward endo-, ecto-, and mesodermal cell lineages. In contrast, Oct4neg MAPC exhibit less broad differentiation capacity, and, importantly, recent extensive characterization has revealed that such Oct4neg mouse MAPC most closely resembles the MAPC that can be isolated from human bone marrow. Our initial studies were directed at characterizing Oct4pos mouse MAPC, after which we focused on the study of Oct4neg mouse MAPC, in comparison with mouse MSC. In in vitro studies we observed that it is a general feature of mouse MAPC to be hypo-immunogenic for T-cells and susceptible for NK cell-mediated cytotoxicity; this supports the safety of their use as a third-party cell-source for clinical applications, but implies that in vivo survival may be limited as a result of by NK cell killing. We found that mouse MAPC are able to suppress alloreactive T-cell proliferation in in vitro cellular assays and in vivo, in a local GvHD assay. Importantly, however, we found that Oct4pos mouse MAPC under selected circumstances exhibit immune-stimulatory effects in vitro, whereas Oct4neg mouse MAPC consistently exhibit T-cell suppressive effects that are similarly efficient to those exerted by mouse MSC. These data indicated that when interpreting mouse data, cell-line dependent variation should be taken into account, and this was further supported by the fact that the single other report on Oct4-expressing mouse MAPC, that was published during course of our investigations, did not report bimodal immunomodulatory effects. Our data indicate that the use of Oct4pos MAPC may hold safety concerns for in vivo application. In contrast, the data obtained with the Oct4neg clone support the exploration of Oct4-negative human MAPC as an alternative to MSC for the treatment and/or prevention of GvHD.In the second part of this work, we addressed the immunoregulatory role of myeloid precursor cells that have been shown to expand endogenously as a bystander phenomenon in transplant protocols. In particular we aimed to determine whether these correspond to typical MDSC, until now best characterized in the field of tumor immunity. Here, MDSC refer to a bone marrow-derived population of myeloid precursor cells at different stages of differentiation, which play a critical role in the mechanisms used by solid tumors to escape T-cell immunity. Importantly, increasing evidence in tumor-bearing mice and cancer patients underlines the importance of studying subtypes of MDSC and suppressive mechanisms, since these were found to be different. Hypothesizing that endogenously occurring MDSC may play a role in controlling post-transplant GvH reactivity, we explored the presence and function of MDSC and subtypes of MDSC in an experimental and clinical setting of allogeneic HSCT. We first performed a detailed phenotypic study of myeloid precursor cells and subsets in a C57BL/6→F1 hybrid mouse model where we previously showed myeloid precursor cells to expand in the recipient of allogeneic stem cell grafts, and investigated in vitro and in vivo function. Interestingly, in this model, as well as in an irradiation bone marrow transplant model described previously, we had already showed that such expansion coincided with a time-frame wherein DLI mediates strong LH-GvHR but no fatal GvHD. Hence we postulated that these myeloid precursor cells may regulate the activity of allogeneic T-cells derived from the bone marrow and/or DLI graft. Secondly, we investigated the phenotype and function of human myeloid suppressor cells in a clinical setting of allogeneic HSCT, in particular, in the peripheral blood of G-CSF-treated stem cell donors. Such cells have been reported to expand but so far, have been poorly characterized and vaguely described as suppressive CD14+ monocytes and activated granulocytes . Our phenotype and morphology studies revealed that both in the mouse and the human setting the expanded myeloid precursor cell population comprises the typical mononuclear and polymorphonuclear MDSC that have been recently described in tumor-bearing mice and cancer patients.157;158157;158157;158157;158157;158157;158 We found that both MDSC subtypes are capable of regulating T-cell alloreactivity in vitro, and for the mouse model also in vivo in a local GvHD assay. Interestingly, in the mouse model, we found evidence to suggest that these MDSC play a role in modulating the reactivity of adoptively transferred non-tolerant donor T-cells, a situation reminiscent of the clinical practice of DLI. In the mouse model, we started deciphering T-cell suppressive mechanism and found that iNOS is involved in MO-MDSC mediated suppression, which is conform reports on tumor-associated MDSC subtypes. However, we were not able to identify mediators involved in PMN-MDSC-mediated suppression.Data from this study may serve as a starting point to investigate the role of endogenously expanding MDSC -occurring either within the stem cell graft or in the stem cell recipient- in influencing the reactivity of donor T-cells and the risk of GvHD in the peri-transplant period.nrpages: 134status: publishe