The immune response is a major barrier to the successful transplantation of organs and tissues required in the treatment of many human diseases. Although the field of tissue engineering was created to address the shortage of human organs and tissues, the immune response remains a substantial challenge, impeding the development of allogeneic biological substitutes to repair, replace and regenerate tissues. Specifically, the T cell mediated immune response initiated through the recognition of cell surface Major histocompatibility complex Class I (MHCI) molecules is the primary cause of acute allograft rejection. In nature, viruses have evolved many mechanisms to exploit weaknesses of the T cell response to evade detection. Viral mechanisms to modulate the MHCI molecule can be effectively applied to allogeneic cells in a tissue-engineered construct to evade detection by CD8+ Cytotoxic T Lympocytes (CTLs) and Natural Killer (NK) cells of the immune system. We demonstrate the successful application of a retroviral vector to over-express the Kaposi's sarcoma-associated herpesvirus (KSHV) immunomodulatory protein, MIR2, in human monocyte-like leukemia cells to differentially downregulate cell surface MHCI, ICAM-1 and B7-2 molecules. We also developed a novel flow cytometry-based cytotoxicity assay to demonstrate that this differential downregulation of immunoactive molecules has the functional effect of significantly reducing CTL-mediated cytotoxicity, without altering NK-mediated cytotoxicity. We believe that this approach provides a potential solution to circumvent the acute immune rejection of allografts in vivo, and can also lead to the development of "universal" donor cells for tissue engineering applications that will not require anti-rejection drugs. / Thesis / Master of Applied Science (MASc)

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Etude des Treg dans la physiopathologie du syndrome de Wiskott-Aldrich et étude de la reconstitution des Treg dans les greffes de cellules souches hématopoïétiques après un conditionnement nonmyéloablateur.

It has been shown that when implanted individually, both cells and biomaterials elicit biological responses. Implanted cells are often destroyed by the host's immune system, while biomaterials activate foreign body reactions which can result in inflammation and fibrotic encapsulation. However, when implanted simultaneously, the inflammatory responses to the biomaterial component can alter the immune responses to the cellular component. The experiments described in this thesis were designed to characterize the effect of different biomaterials on adaptive immune responses towards an allogeneic challenge. Balb/c splenocytes were challenged with irradiated allogeneic L929 cells, and treated with different biomaterials. Alterations in adaptive immune responses were quantified by T cell proliferation and cytokine release (i.e. IL-1(beta), IL-4, IL-12, and IFN-(gamma)). The roles various cell types played in first set responses were investigated. Experimental results indicated that biomaterials had a significant influence on nonspecific proliferation of splenocytes. In particular, analysis of the degree to which biomaterials affected specific proliferation indicated that the soluble alginate treatment significantly increased proliferation differences when compared to the control. However, biomaterials neither significantly affected specific splenocyte proliferation to an allogeneic challenge, nor the profile of secreted cytokines. To elucidate this response, alginate-treated splenocytes were depleted of adherent macrophages, CD4+ cells or CD8+ cells. Within non-challenged mixtures, CD4+ depletion had the most obvious effect. These results were supported by the non-depleted challenges, and indicated the direct influence biomaterials on CD4+ T cell proliferation. / Thesis / Master of Applied Science (MASc)

Immunomodulatory drugs (IMiDs) could enhance both direct anti-tumour and graft-versus-tumour effects after allogeneic haematopoietic stem cell transplantation (AHSCT). However, clinical experience with IMiDs after AHSCT using adult peripheral blood (APB) as a stem-cell source has been limited by graft-versus-host disease. Characterization of the mechanisms by which IMIDs modulate alloresponses of T cells and identification of differential effects on T cells from different cell sources could facilitate more effective use of these drugs in the setting of AHSCT. Using in vitro modelling, multi-parameter flow cytometry and gene expression analysis, I have determined the impact of the widely used IMiD lenalidomide on alloresponses of APB and umbilical cord blood (UCB)-derived T cells. Lenalidomide-treatment potentiates net alloproliferation of APB-derived T cells by selectively enhancing proliferation of CD8+ T cells. These CD8+ T cells have enhanced effector memory differentiation, are enriched for polyfunctional effectors, have enhanced direct-cytotoxicity against heamatopoietic target-cells and have a distinct gene expression profile with altered expression of key immunoregulatory-genes and depletion of cellular ikaros. Importantly, while effects on CD8+ T cells derived from UCB are similar, lenalidomide has contrasting effects on allospecific proliferation of APB and UCB-derived CD4+ T cells. While lenalidomide-treatment has no effect on alloproliferation of APB-derived CD4+ T cells, it reduces alloproliferation of UCB-derived CD4+ T cells. The reduction in UCB-derived CD4+ T cell alloproliferation is accompanied by selective expansion of CD4+CD25+FOXP3+ regulatory T cells (Treg), resulting in an overall reduction in UCB-derived T cell alloproliferation. These findings demonstrate that lenalidomide has a differential impact on alloresponses of T cells from different cell sources; alloresponses of APB-derived T cells are increased via selective expansion of polyfunctional CD8+ effectors, while alloresponses of UCB-derived T cells are limited by expansion of tolerogenic Treg. These findings have important implications for the future use of IMiDs in the setting of AHSCT.

Through the recruitment of immunologic mechanisms, allogeneic hematopoeitic cell transplantation (HCT) has been establiched as a curative treatment for various hematologic diseases. The most convenient source to obtain hematopoietic progenitor cells are peripheral blood stem cells (PBSCs) which are harvested from the donor via leukapheresis after mobilization with granulocyte-colony stimulating factors. With the introduction of reduced intensity condition (RIC), the curative potential of allogeneic HCT became accessible to older and/or frail patients otherwise ineligible for HCT. However, new challenges arise as the increasing age of patients is inevitably accompanied by a comparable increase in the age of donors. Safety considerations of collecting PBSCs might attain new dimensions. Data to potential risks in elderly donors are lacking. Moreover, the impact of donor’s age on the feasibility of PBSCs collection and on the quality of the harvest in terms of stem cells (CD34+) and natural killer (NK)-cells has not been studied. It is also unknown whether PBSCs obtained from donors above 50 years would negatively influence engraftment or the incidence of graft-versus-host disease (GVHD) in the recipient. These questions were explored in a retrospective study including 167 recipients of an allogeneic HCT (52.7% after RIC) from a matched related sibling. Median donors’ age was 47 years [67 (40%) donors were > 50 years including 34 donors > 60 years]. Safety of mobilization and leukapheresis was age independent. Adequate PBSCs were collected from all donors though a higher CD34+-cell count was seen in donors < 50 years (p<0.0005), whereas harvests from donors > 60 years contained a higher NK-cell count (p=0.003). Engraftment in the recipient occurred after a median of 12 days and was not affected by an advanced donor age. Similarly, a higher incidence of GVHD was not seen in recipients of harvests from older donors. For the first time, we show that donor’s age, even beyond 60 years, does not preclude successful collection of PBSCs from siblings, does not jeopardize the short-term safety of the donor, and is not associated with deleterious sequels for the recipient in terms of engraftment or GVHD. As NK-cells have been implicated in the suppression of GVHD, and the mediation of a graft versus leukemia effect, the impact of the higher number of NK-cells in harvests from elderly donors on relapse of hematologic malignancies in the recipient warrants further studies.

Class of 2016 Abstract / Objectives: The purpose of this study was to evaluate if fluconazole 200 mg is an acceptable alternative to the fluconazole 400 mg for fungal prophylaxis in allogenic hematopoietic stem cell patients. Lower fluconazole doses will decrease cost of therapy and may reduce adverse events associated with higher doses. Methods: This study was a retrospective chart review conducted at the Arizona Cancer Center. A total of 58 patients qualified for the study. Primary endpoints were number of days on fluconazole 200 mg and type and number of fungal infections that occurred within 1 year post transplant. Results: Out of the fifty-eight patients who qualified for the study, only eight patients had a breakthrough fungal infection while on 200 mg (13.7%) after one year. Three of those eight were identified as having systemic fungal infections (5.2%). Conclusions: Fluconazole 200 mg is a reasonable low-cost and low side effect alternative to fluconazole 400 mg for antifungal prophylaxis in allogenic hematopoietic stem cell patients.

Through the recruitment of immunologic mechanisms, allogeneic hematopoeitic cell transplantation (HCT) has been establiched as a curative treatment for various hematologic diseases. The most convenient source to obtain hematopoietic progenitor cells are peripheral blood stem cells (PBSCs) which are harvested from the donor via leukapheresis after mobilization with granulocyte-colony stimulating factors. With the introduction of reduced intensity condition (RIC), the curative potential of allogeneic HCT became accessible to older and/or frail patients otherwise ineligible for HCT. However, new challenges arise as the increasing age of patients is inevitably accompanied by a comparable increase in the age of donors. Safety considerations of collecting PBSCs might attain new dimensions. Data to potential risks in elderly donors are lacking. Moreover, the impact of donor’s age on the feasibility of PBSCs collection and on the quality of the harvest in terms of stem cells (CD34+) and natural killer (NK)-cells has not been studied. It is also unknown whether PBSCs obtained from donors above 50 years would negatively influence engraftment or the incidence of graft-versus-host disease (GVHD) in the recipient. These questions were explored in a retrospective study including 167 recipients of an allogeneic HCT (52.7% after RIC) from a matched related sibling. Median donors’ age was 47 years [67 (40%) donors were > 50 years including 34 donors > 60 years]. Safety of mobilization and leukapheresis was age independent. Adequate PBSCs were collected from all donors though a higher CD34+-cell count was seen in donors < 50 years (p<0.0005), whereas harvests from donors > 60 years contained a higher NK-cell count (p=0.003). Engraftment in the recipient occurred after a median of 12 days and was not affected by an advanced donor age. Similarly, a higher incidence of GVHD was not seen in recipients of harvests from older donors. For the first time, we show that donor’s age, even beyond 60 years, does not preclude successful collection of PBSCs from siblings, does not jeopardize the short-term safety of the donor, and is not associated with deleterious sequels for the recipient in terms of engraftment or GVHD. As NK-cells have been implicated in the suppression of GVHD, and the mediation of a graft versus leukemia effect, the impact of the higher number of NK-cells in harvests from elderly donors on relapse of hematologic malignancies in the recipient warrants further studies.

A 6 year old boy developed a fatal, rapidly progressive encephalopathy 5 months after a matched unrelated cord blood transplant. Autopsy findings revealed spongiform changes in his brain. The clinical course of this child's illness had many findings consistent with that of a transmissible spongiform encephalopathy (TSE). Pre-mortem and post-mortem studies failed to definitively determine an etiology. Spongiform encephalopathies include the TSEs and mitochondrial encephalopathies. Both should be considered in a post-hematopoietic stem cell transplant patient who develops a progressive encephalopathy when more common etiologies are not found.

Im Rahmen dieser Arbeit wurden 72 pädiatrische Patienten, die eine allogene Stammzelltransplantation erhielten, auf die Spender- und Empfänger-Zytokinpolymorphismen TNF-α, TGF-β1, IL-10, IL-6 und IFN-γ untersucht. Die Proben wurden mittels DNA-Extraktion, sequenzspezifischer PCR und Gelelektrophorese analysiert und auf deren Einfluss auf die Entstehung und den Verlauf maligner Erkrankungen sowie auf die Entstehung einer GvHD untersucht. / Within the scope of this work, 72 pediatric patients who received allogeneic stem cell transplantations were examined for the donor and recipient cytokine polymorphisms TNF-α, TGF-β1, IL-10, IL-6 and IFN-γ. We analyzed the samples by means of DNA extraction, sequence-specific PCR and gel electrophoresis and investigated their influence on the development and course of malignant diseases as well as on the development of a GvHD.