O transplante de células-tronco hematopoéticas alogênico (alo-TCTH) é uma opção de tratamento para uma variedade de doenças neoplásicas e não neoplásicas, principalmente de origem hematológica sendo doença do enxerto-contra-hospedeiro (DECH) a sua principal complicação. As células Natural Killer (NK) são os primeiros linfócitos a se recuperarem após o TCTH. Além da capacidade de promover o efeito enxerto-versus-leucemia (EVL), as células NK do doador parecem capazes de promover a pega do enxerto e de prevenir o desenvolvimento da DECH. As células NK compreendem aproximadamente 10% dos linfócitos do sangue periférico e são caracterizadas fenotipicamente pela expressão do antígeno de superfície CD56 (CD, cluster of differentiation) e pela ausência de CD3 (CD56+CD3-). O subtipo de células NK CD56dim (baixa densidade do antígeno) é naturalmente mais citotóxico que o subtipo CD56bright (alta densidade do antígeno) o qual é caracterizado pela capacidade de produção de citocinas. Com base nisso, o objetivo do trabalho é avaliar a presença de células NK nos dias 7, 14, 21 e 28 após o TCTH alogênico e autólogo, caracterizando sua frequência, seu imunofenótipo e a sua capacidade de produzir fatores de crescimento hematopoético e citocinas relacionadas. / Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an option of treatment for a variety of neoplastic and non-neoplastic diseases and graft-versus-host disease (GVHD) is its main complication. Natural Killer cells (NK) are the first lymphocytes to recover after HSCT. In addition to the ability to promote graft versus leukemia effect (GVL), donor NK cells appear to be capable of promoting engraftment and preventing the development of GVHD. NK cells comprise approximately 10% of peripheral blood lymphocytes and are characterized phenotypically by the expression of the CD56 surface antigen and absence of CD3 (CD56 + CD3-). The CD56dim (low density of antigen) NK cell subtype is naturally more cytotoxic than the CD56bright (high density of antigen) subtype which is characterized by the ability to produce cytokines. Based on this, the objective of the study is to evaluate the presence of NK cells on days 7, 14, 21 and 28 after allogeneic and autologous HSCT, characterizing their frequency, their immunophenotype and their capacity to produce hematopoietic growth factors and related cytokines.

O transplante de células-tronco hematopoéticas alogênico (alo-TCTH) é uma opção de tratamento para uma variedade de doenças neoplásicas e não neoplásicas, principalmente de origem hematológica sendo doença do enxerto-contra-hospedeiro (DECH) a sua principal complicação. As células Natural Killer (NK) são os primeiros linfócitos a se recuperarem após o TCTH. Além da capacidade de promover o efeito enxerto-versus-leucemia (EVL), as células NK do doador parecem capazes de promover a pega do enxerto e de prevenir o desenvolvimento da DECH. As células NK compreendem aproximadamente 10% dos linfócitos do sangue periférico e são caracterizadas fenotipicamente pela expressão do antígeno de superfície CD56 (CD, cluster of differentiation) e pela ausência de CD3 (CD56+CD3-). O subtipo de células NK CD56dim (baixa densidade do antígeno) é naturalmente mais citotóxico que o subtipo CD56bright (alta densidade do antígeno) o qual é caracterizado pela capacidade de produção de citocinas. Com base nisso, o objetivo do trabalho é avaliar a presença de células NK nos dias 7, 14, 21 e 28 após o TCTH alogênico e autólogo, caracterizando sua frequência, seu imunofenótipo e a sua capacidade de produzir fatores de crescimento hematopoético e citocinas relacionadas. / Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an option of treatment for a variety of neoplastic and non-neoplastic diseases and graft-versus-host disease (GVHD) is its main complication. Natural Killer cells (NK) are the first lymphocytes to recover after HSCT. In addition to the ability to promote graft versus leukemia effect (GVL), donor NK cells appear to be capable of promoting engraftment and preventing the development of GVHD. NK cells comprise approximately 10% of peripheral blood lymphocytes and are characterized phenotypically by the expression of the CD56 surface antigen and absence of CD3 (CD56 + CD3-). The CD56dim (low density of antigen) NK cell subtype is naturally more cytotoxic than the CD56bright (high density of antigen) subtype which is characterized by the ability to produce cytokines. Based on this, the objective of the study is to evaluate the presence of NK cells on days 7, 14, 21 and 28 after allogeneic and autologous HSCT, characterizing their frequency, their immunophenotype and their capacity to produce hematopoietic growth factors and related cytokines.

O transplante de células-tronco hematopoéticas alogênico (alo-TCTH) é uma opção de tratamento para uma variedade de doenças neoplásicas e não neoplásicas, principalmente de origem hematológica sendo doença do enxerto-contra-hospedeiro (DECH) a sua principal complicação. As células Natural Killer (NK) são os primeiros linfócitos a se recuperarem após o TCTH. Além da capacidade de promover o efeito enxerto-versus-leucemia (EVL), as células NK do doador parecem capazes de promover a pega do enxerto e de prevenir o desenvolvimento da DECH. As células NK compreendem aproximadamente 10% dos linfócitos do sangue periférico e são caracterizadas fenotipicamente pela expressão do antígeno de superfície CD56 (CD, cluster of differentiation) e pela ausência de CD3 (CD56+CD3-). O subtipo de células NK CD56dim (baixa densidade do antígeno) é naturalmente mais citotóxico que o subtipo CD56bright (alta densidade do antígeno) o qual é caracterizado pela capacidade de produção de citocinas. Com base nisso, o objetivo do trabalho é avaliar a presença de células NK nos dias 7, 14, 21 e 28 após o TCTH alogênico e autólogo, caracterizando sua frequência, seu imunofenótipo e a sua capacidade de produzir fatores de crescimento hematopoético e citocinas relacionadas. / Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an option of treatment for a variety of neoplastic and non-neoplastic diseases and graft-versus-host disease (GVHD) is its main complication. Natural Killer cells (NK) are the first lymphocytes to recover after HSCT. In addition to the ability to promote graft versus leukemia effect (GVL), donor NK cells appear to be capable of promoting engraftment and preventing the development of GVHD. NK cells comprise approximately 10% of peripheral blood lymphocytes and are characterized phenotypically by the expression of the CD56 surface antigen and absence of CD3 (CD56 + CD3-). The CD56dim (low density of antigen) NK cell subtype is naturally more cytotoxic than the CD56bright (high density of antigen) subtype which is characterized by the ability to produce cytokines. Based on this, the objective of the study is to evaluate the presence of NK cells on days 7, 14, 21 and 28 after allogeneic and autologous HSCT, characterizing their frequency, their immunophenotype and their capacity to produce hematopoietic growth factors and related cytokines.

Die akute Graft-versus-Host-Krankheit (GvHD) ist eine der Hauptkomplikationen nach einer allogenen hämatopoetischen Stammzelltransplantation, die die Effizienz der Therapie und deren Einsatz limitiert. Aktuelle Präventionsstrategien und Therapien beinhalten systemisch wirkende Immunsuppressiva. Diese haben oft zahlreiche Nebenwirkungen und erhöhen die Rate von Tumorrezidiven und schweren Infektionen. Therapierefraktäre Verläufe der GvHD sind häufig und können mit einer schlechten Prognose vergesellschaftet sein. Neuartige Präventions- und Behandlungsansätze sind daher Gegenstand intensiver Forschung. Eine zentrale Rolle in der Pathogenese der akuten GvHD spielen CD4+ T Zellen. Alloreaktive T Zellen vermitteln eine proinflammatorische Immunantwort, produzieren entsprechende Zytokine und aktivieren weitere Effektorzellen. Diese Immunkaskade induziert eine systemische Entzündungsreaktion und führt zu Organ- und Gewebsschädigung. Der spezifischen Modulation dieser alloreaktiven CD4+ T Zellen hin zu einer Toleranzentwicklung gegenüber dem Empfängergewebe wird eine große Bedeutung in der Entwicklung innovativer GvHD-Präventionsstrategien beigemessen. Es konnte in murinen Modellen gezeigt werden, dass die ex vivo-Behandlung eines allogenen bzw. xenogenen Transplantats mit dem CD4-gerichteten nicht-depletierenden Antikörper MAX.16H5 zu einer verminderten GvH-Reaktion führte. Eine Beeinträchtigung des Graft-versus-Leukämie-Effekts war nicht nachweisbar. Toleranzinduktionen durch monoklonale Antikörper gegen Oberflächenrezeptoren von Immunzellen im Rahmen von Autoimmunerkrankungen und GvHD werden in zahlreichen Studien untersucht. Darüber hinaus ist bekannt, dass in der GvHD-Pathogenese bestimmte microRNAs, 17 bis 25 Nukleotide lange, nicht kodierende, einzelsträngige RNA-Moleküle, eine Schlüsselrolle spielen. Die molekularen Mechanismen, die der Toleranzentwicklung des MAX.16H5 Antikörpers zugrunde liegen, sind jedoch nicht abschließend geklärt. Daher war es Ziel der vorliegenden Doktorarbeit (i) ein in vitro Stimulationsmodell für weitergehende Untersuchungen am MAX.16H5 Antikörper zu etablieren und (ii) durch Quantifizierung der microRNA-Expression unter T Zellstimulierung zur Aufklärung von molekularen Mechanismen der Toleranzinduktion des Antikörpers beizutragen. Das etablierte in vitro Stimulationsmodell diente zur Analyse und Phänotypisierung von CD4+ T Zellen nach Antikörperinkubation und Stimulation. Es konnte gezeigt werden, dass eine MAX.16H5-Behandlung zu einer verminderten Aktivierung (CD25-Expression) von T Zellen nach 24 h und 72 h in Kokultur mit murinen Milzzellen bei einem 10:1 bzw. 8,2:1 Verhältnis (humane CD4+ T Zellen zu murinen Milzzellen) führte. Dieses Modell kann als Grundlage für weitere in vitro Studien dienen, um Prozesse der Toleranzinduktion durch monoklonale Antikörper zu untersuchen, und liefert einen wichtigen Beitrag für die präklinische Analyse des MAX.16H5 Antikörpers im Hinblick auf die Entwicklung funktioneller Tests. Weiterhin wurde in dieser Arbeit die Expression von microRNAs in CD4+ T Zellen nach Bindung des MAX.16H5 Antikörpers untersucht. Hierzu wurden humane CD4+ T Zellen mit dem MAX.16H5 Antikörper behandelt und (i) ohne Stimulation, (ii) mit murinen Milzzellen bzw. (iii) mit Phytohämagglutinin inkubiert. Das microRNA-Expressionsprofil wurde mit Next Generation Sequencing bestimmt. In dieser Screeninganalyse wurden zahlreiche microRNAs gefunden, die unter den verschiedenen Stimulationsbedingungen nach MAX.16H5-Inkubation differentiell exprimiert waren. Die Expression der miR-18a-3p und miR-598-3p wurde anschließend mit qPCR näher untersucht. Eine statistisch signifikante Regulation konnte für miR-598-3p nach 72 h Inkubation nachgewiesen werden. Es ist der bisher einzige Hinweis auf einen molekularen Effekt der MAX.16H5-Behandlung auf CD4+ T Zellen. Ob dieser Unterschied funktioneller Natur, im Sinne eines Toleranz-induzierenden Phänotyps ist, werden zukünftige Studien zeigen. Zudem wird aktuell die miR-598-3p als potenzieller Biomarker für den Nachweis einer erfolgreichen Inkubation mit MAX.16H5 untersucht, was für zukünftige klinische Studien von großer Wichtigkeit ist.:1. Einleitung 1 1.1 Literaturübersicht 1 1.1.1 Die hämatopoetische Stammzelltransplantation 1 1.1.2 Die allogene Stammzelltransplantation 2 1.1.3 Die Graft-versus-Host-Krankheit 5 1.1.4 Die Prävention und Behandlung der akuten GvHD 8 1.1.5 Neue Strategien zur GvHD-Behandlung und -Prävention 10 1.1.6 Der Graft-versus-Leukämie-Effekt 13 1.1.7 Der CD4-gerichtete MAX.16H5 Antikörper zur Prävention der GvHD 14 1.1.8 Die Toleranzinduktion durch monoklonale Antikörper 16 1.1.9 Die microRNA 19 1.1.10 Die Rolle von miRNAs in der GvHD 20 1.1.11 Die Rationale für die Untersuchung von miR-18a-3p 21 1.1.12 Die Rationale für die Untersuchung von miR-598-3p 22 2 Fragestellung und Experimentdesign 23 3 Material und Methoden 25 3.1 Materialien 25 3.1.1 Verwendete Zellen 25 3.1.2 Geräte 25 3.1.3 Chemikalien, Medien und Reagenzien 26 3.1.4 Verbrauchsmaterialien 27 3.1.5 Oligonukleotide 28 3.1.6 Antikörper 28 3.1.6.1 Antikörper für Durchflusszytometrie 28 3.1.6.2 Antikörper für Zellinkubation 29 3.2 Methoden 29 3.2.1 Schematische Darstellung der methodischen Arbeitsschritte 29 3.2.2 Zellkulturarbeiten 31 3.2.2.1 Auftauen von Zellen 31 3.2.2.2 Konservierung von Zellen 31 3.2.2.3 Bestimmung der Zellzahl von PBMCs und Splenozyten 32 3.2.3 Isolation humaner PBMCs aus Spenderblut 32 3.2.4 Isolation muriner Milzzellen 33 3.2.5 Isolation humaner CD4+ T Zellen und MACS-Separation humaner und muriner Zellen 33 3.2.6 Inkubation mit dem murinem MAX.16H5 IgG1 Antikörper 35 3.2.7 Inkubation humaner CD4+ T Zellen 35 3.2.8 Durchflusszytometrie 36 3.2.9 RNA-Isolation 37 3.2.10 RNA-Fällung 38 3.2.11 Next Generation Sequencing von miRNAs 38 3.2.12 Next Generation Sequencing – Analyseschema 41 3.2.13 Reverse Transkription und qPCR 41 3.2.13.1 Reverse Transkription 42 3.2.13.2 qPCR 43 3.2.14 Statistische Analyse, Tabellen und Abbildungen 44 4. Ergebnisse 46 4.1 Etablierung eines in vitro Stimulationsmodells zur Analyse humaner CD4+ T Zellen nach Antikörperinkubation 46 4.2 Next Generation Sequencing zur miRNA-Expressionsanalyse in CD4+ T Zellen nach MAX.16H5 IgG1-Inkubation 50 4.2.1 FACS-Analyse isolierter CD4+ T Zellen vor und nach Antikörperinkubation 50 4.2.2 Separation muriner Milzzellen von humanen CD4+ T Zellen 53 4.2.3 NGS-miRNA-Expressionsanalyse in stimulierten und unstimulierten CD4+ T Zellen nach 72 h Inkubation 55 4.2.3.1 MiRNA-Expression nach MAX.16H5 IgG1-Inkubation und PHA-Stimulation 57 4.2.3.2 MiRNA-Expression nach MAX.16H5 IgG1-Inkubation und Stimulation mit murinen Milzzellen 59 4.2.3.3 MiRNA-Expression nach MAX.16H5 IgG1-Inkubation und ohne Stimulation 61 4.2.4 Zusammenfassung der Ergebnisse der NGS-Analyse 63 4.3 FACS-Analyse von CD4+ T Zellen nach 24 h und 72 h Inkubation 64 4.3.1 FACS-Analyse von CD4+ T Zellen nach 24 h Inkubation 64 4.3.2 FACS-Analyse von CD4+ T Zellen nach 72 h Inkubation 67 4.4 Validierung der Kandidaten-miRNA mittels qPCR 72 4.4.1 Expression von miR-18a-3p und miR-598-3p nach MAX.16H5 IgG1-Inkubation in CD4+ T Zellen nach 72 h 72 4.4.1.1 FACS-Analyse isolierter CD4+ T Zellen vor und nach Antikörperinkubation 72 4.4.1.2 FACS-Analyse der CD4+ T Zellen nach 72 h Inkubation 74 4.4.1.3 Separation muriner Milzzellen von humanen CD4+ T Zellen 78 4.4.1.4 Expression von miR-18a-3p und miR-598-3p 80 5. Diskussion 85 5.1 Die Expression von miR-18a-3p und der miR-17–92 Cluster 86 5.2 Die Expression von miR-598-3p 88 5.3 Die Differentielle Expression weiterer miRNAs 90 5.3.1 miR-223 90 5.3.2 Let-7d 91 5.3.3 miR-21 91 5.3.4 miR-181c 91 5.3.5 miR-10a 92 5.3.6 miR-150 92 5.3.7 miR-199a 92 5.3.8 miR-155 93 5.4 Die miRNA-Expressionsanalyse mit Next Generation Sequencing 93 5.5 Die Etablierung eines in vitro Stimulationsmodells 96 5.6 Verminderte T Zell-Aktivierung durch MAX.16H5 IgG1 98 6 Zusammenfassung der Arbeit 101 7 Literaturverzeichnis 104 A Erklärung über die eigenständige Abfassung der Arbeit 129 B Erklärung über die Vorbehaltlichkeit der Verfahrenseröffnung zur Verleihung des Titels Dr. med. 130 C Darstellung des wissenschaftlichen Werdegangs 131 D Danksagung 133

Allogeneic Hematopoietic stem cell transplants (HSCT) are used for the treatment of bone marrow aplasias. Allogeneic HSCT is performed by treating the patient with chemotherapy drugs and irradiation and then transplanting hematopoietic stem cells from a healthy donor to restore the immune system and hematopoietic cells. Allogeneic HSCTs has the added benefit of the graft vs leukemia effect (GvL), whereby donor allogeneic T cells are able to mount immune responses against any residual cancer cells. However, alloreactivity towards the mismatched minor and major histocompatibility antigens the patient's healthy tissues leads to graft vs host disease (GvHD). This process is also mediated by Macrophages, Dendritic cells, B cells. Furthermore, a decrease in the number of NK, B, and T regulatory cells exacerbates GvHD. This leads to a state of systemic inflammation, tissue damage and multiorgan fibrosis. Current therapies designed to suppress the immune system have been shown to be efficacious in preventing GvHD but patients become susceptible to infection or experience cancer relapse through the elimination of the GvL response as well. In this thesis, we explore two strategies for targeting T cell activation in two mouse models of GvHD. In the first model, we examined the contribution of donor-derived complement C5 on the induction GvHD. We observed that recipient mice were only protected from GvHD when donor cells were deficient for complement protein C5. Our second strategy involves selective targeting of alloreactive T cells using peptide immunotherapy. For this approach, we first developed a humanized mouse model of GvHD whereby cells from donor mice expressing human class II HLA were reconstituted into recipient mice expressing human class I HLA. We then tested peptide immunotherapy using peptides derived from the human class I HLA. Our initial results were inconclusive and require further optimization. / Thesis / Doctor of Philosophy (PhD) / Graft vs Host Disease is an unwanted side effect of mismatched bone marrow transplant. Donor T cells recognize and attack mismatched tissues of the recipient and this leads to systemic inflammation and tissue scarring. Current treatments primarily target T-cell activation by suppressing the immune system, however, this leaves the patients susceptible to recurrent infections. In this thesis we describe the creation of two mouse models of Graft vs Host Disease and then examine two ways of specifically targeting donor T cell activation that is designed not to affect normal immune responses.

La réaction du greffon contre l’hôte (GvH) est responsable d’un grand taux de morbidité et de mortalité chez les patients recevant des greffes de cellules souches (GCSH) allogéniques. Dans ce contexte, les cellules T régulatrices sont largement étudiées et semblent avoir un grand potentiel d’utilisation dans le domaine de la thérapie cellulaire de la GvH. Parmi les populations cellulaires T régulatrices, les lymphocytes T CD4-CD8- TCRαβ+ « Doubles-Négatifs » (DN), qui ne représentent que 1-3% des lymphocytes T, ont été décrits. Ces cellules ont des propriétés inhibitrices de la réponse immunitaire qui s’avèrent spécifiques aux antigènes auxquels elles ont préalablement été exposées. La répression de la réponse immunitaire par les cellules T DN régulatrices semble être un mécanisme important impliqué dans l’induction de la tolérance aux allo-antigènes. De plus, ces cellules confèrent une tolérance immunitaire dans des modèles de greffes allogéniques et xénogéniques. En effet, ces cellules ont la capacité d’inhiber la réaction contre un allo-antigène auquel elles ont été exposées, sans inhiber la réaction contre un allo-antigène inconnu. Les cellules T DN ont été isolées et caractérisées chez l’homme où elles ont la capacité d’interagir avec des cellules présentatrices d’antigènes (APCs) par un contact cellulaire, comme chez la souris. Cependant, leur capacité immunomodulatrice reste inconnue chez l’humain. Notre objectif consistait donc principalement à étudier le rôle et le mécanisme d’action des cellules T DN régulatrices humaines in vitro, en étudiant leur capacité à inhiber une réaction lymphocytaire mixte (MLR). Nous avons montré que les cellules T DN stimulées par un allo-antigène donné inhibent des cellules syngéniques effectrices dirigées contre ce même alloantigène mais n’inhibent pas des cellules syngéniques effectrices dirigées contre un autre alloantigène, démontrant ainsi la spécificité aux antigènes de ces cellules. De plus, les T DN non stimulées par un allo-antigène n’ont pas de rôle inhibiteur. Cependant, durant cette inhibition, nous n’observons pas de modulation de l’expression des marqueurs d’activation et d’induction de l’apoptose. Afin d’étudier le mécanisme d’action des cellules T DN, nous avons mesuré l’expression intracellulaire de la granzyme B. Les résultats démontrent que les cellules T DN stimulées expriment un niveau significativement plus élevé de granzyme B que les cellules T DN non-stimulées par l’allo-antigène. Ceci suggère que l’immunosuppression induite par les cellules T DN stimulées pourrait passer par la voie granzyme B. Le mécanisme utilisé par ces cellules reste à être confirmé par nos futures expériences. / Graft-versus-Host Disease (GvHD) is a major cause of morbidity and mortality in patients receiving an allogeneic Hematopoietic Stem Cell Transplantation (HSCT). Many regulatory T cell populations have been studied and shown to have immunosuppressive properties in GvHD. Among these populations, Double Negative CD4-CD8-TCRαβ+ regulatory T cells (DN T) have been described. These cells represent 1-3% of all T cell lymphocytes and are known to have antigen-specific inhibitory functions of the immune response. The suppression of an immune response by DN T cells seems to be an important mechanism involved in the induction of tolerance to allo-antigens. Moreover, these cells also confer immune tolerance in models of allogeneic and xenogenic grafts. DN T cells have the ability to suppress syngeneic T CD4+ and T CD8+ cells in an antigen-specific manner. Therefore, these DN T cells can inhibit the reaction caused by syngeneic effector cells against a specific alloantigen to which they have been previously exposed. However, they cannot inhibit a reaction directed against an unknown alloantigen. Human DN T cells have been isolated and characterized as cells that have the capacity to interact with APCs by cell-to-cell contact, just like in mice. However, their immunomodulatory properties are still unknown in humans. The goal of our project was to study the role and immunomodulatory functions of human DN T cells in Mixed Lymphocyte Reactions (MLR). The MLRs have allowed us to demonstrate that DN T cells, after having been stimulated by an allo-antigen, have an antigen-specific inhibitory function towards the syngeneic effector cells reacting against the same alloantigen that they have been exposed to. Interestingly, they do not inhibit the reaction of these effector cells against an unknown alloantigen. However, stimulated DN T cells did not modulate the expression of the activation markers expressed by the effector cells and did not give a death signal to these cells either. Moreover, we also wanted to study how DN T cells have an immunosuppressive activity. Therefore, we compared the expression of Granzyme B in stimulated versus non-stimulated cells. Our results suggest that DN T cells may use the Granzyme B pathway to immunosuppress the effector cells. In conclusion, our results demonstrate that DN T cells have an antigen-specific inhibitory function. The mechanism used by these DN T cells remains to be confirmed with our future experiments.

Survival of patients with poor prognosis or relapsed haematopoietic malignancies can be markedly improved by allogeneic haematopoietic stem cell transplantation (HSCT). HSCT reconstitutes the immune and haematopoietic systems after myeloablative conditioning and inhibits the recurrence of the malignancy by a graft-versus-leukaemia (GVL) response mediated by donor T cells. However, significant post-transplant complications such as graft-versus-host disease (GVHD) continue to plague the event-free survival of this curative procedure. GVHD is facilitated by donor T cells that recognise histocompatibility antigens on host antigen presenting cells (APC), such as dendritic cells (DC). Current treatment options for GVHD are focused on these T cells. However, these treatments result in an increased incidence of infection, graft rejection and relapse. A novel means of immunosuppression in GVHD is the use of multi-potent, mesenchymal stromal cells (MSC). MSC are non-immunogenic cells that actively suppress T cell function in vitro, and can resolve steroid-refractory GVHD in the clinic. Despite their use in the clinic, there is a paucity of pre-clinical data. Our aim was to investigate the in vivo efficacy of MSC to control GVHD while maintaining the beneficial GVL effect, and to begin to understand the mechanism by which MSC exert their immunosuppressive effects. We isolated and characterised MSC from murine bone/bone marrow and demonstrated that they suppressed T cell proliferation in vitro, even at low ratios of 1 MSC per 100 T cells. This was true of both donor-derived MSC, and MSC derived from unrelated donors (third party). Importantly, we observed that MSC significantly reduced T cell production of the pro-inflammatory cytokines TNFα and IFNγ in culture supernatants and that IFNγ plays a key role in the ability of MSC to suppress T cell proliferation. In vivo, we examined the effects of donor-derived MSC on GVHD severity and onset in two myeloablative murine models of HSCT. A major histocompatibility complex (MHC)-mismatched donor-recipient pair combination was used as a proof–of-principle model [UBI-GFP/BL6 (H-2b)àBALB/c (H-2d)], and an MHC-matched, minor histocompatibility antigen (miHA) mismatched donor-recipient pair combination was used to mimic MHC-matched sibling transplantation [UBI-GFP/BL6 (H-2b)àBALB.B (H-2b)]. We examined a number of variables related to MSC infusion including timing, dose and route of injection. We found that early post transplant infusion of MSC by the intraperitoneal injection was most effective at delaying death from GVHD, compared to pre-transplant infusion or intravenous injection. Furthermore, we found that the dose of MSC was critical, as infusion of too few MSC was ineffective and infusion of too many MSC exacerbated the development of GVHD. Taken together, these results suggest that timing, dose and route of injection are all important factors to be considered to ensure successful therapeutic outcome. To investigate the in vivo mechanism of action, we conducted timed sacrifice experiments in the MHC-mismatched model to determine if MSC altered cytokine secretion and cellular effectors, such as DC, known to play a key role in GVHD. Despite the fact that MSC given post-HSCT enter an environment full of activated DC and IFNγ levels, by day 3 and 6 post infusion, these activated DC and IFNγ levels are decreased compared to controls or mice infused with MSC pre-transplant (p<0.05). This confirmed our in vitro data that IFNγ played an important role in MSC-mediated immunosuppression. In addition, when we removed a major source of IFNγ production in vivo by administering the T cell depleting antibody KT3 to mice with or without MSC, we found that although T cell depletion prolonged survival, MSC were unable to further enhance this effect. This was also true when MSC were used in combination with the conventional immunosuppressant cyclosporine. Finally, we examined whether the infusion of MSC would compromise the GVL effect. We found that whilst MSC could delay the onset of GVHD, in our model they did not alter the anti-tumour effects of the donor T cells. Overall, we have shown that MSC can delay but not prevent death from GVHD when administered at an appropriate time and dose and that IFNγ is required for MSC-mediated immunosuppression in our model. These data suggest that patients undergoing HSCT should be monitored for IFNγ, and administered MSC when high levels are reached. Whilst MSC may be a promising therapy for patients with severe GVHD, we highlight that further investigation is warranted before MSC are accepted for widespread use in the clinic. The risks and benefits for transplant recipients should be carefully considered before utilising MSC to treat or prevent GVHD.

La réaction du greffon contre l’hôte (GvH) est responsable d’un grand taux de morbidité et de mortalité chez les patients recevant des greffes de cellules souches (GCSH) allogéniques. Dans ce contexte, les cellules T régulatrices sont largement étudiées et semblent avoir un grand potentiel d’utilisation dans le domaine de la thérapie cellulaire de la GvH. Parmi les populations cellulaires T régulatrices, les lymphocytes T CD4-CD8- TCRαβ+ « Doubles-Négatifs » (DN), qui ne représentent que 1-3% des lymphocytes T, ont été décrits. Ces cellules ont des propriétés inhibitrices de la réponse immunitaire qui s’avèrent spécifiques aux antigènes auxquels elles ont préalablement été exposées. La répression de la réponse immunitaire par les cellules T DN régulatrices semble être un mécanisme important impliqué dans l’induction de la tolérance aux allo-antigènes. De plus, ces cellules confèrent une tolérance immunitaire dans des modèles de greffes allogéniques et xénogéniques. En effet, ces cellules ont la capacité d’inhiber la réaction contre un allo-antigène auquel elles ont été exposées, sans inhiber la réaction contre un allo-antigène inconnu. Les cellules T DN ont été isolées et caractérisées chez l’homme où elles ont la capacité d’interagir avec des cellules présentatrices d’antigènes (APCs) par un contact cellulaire, comme chez la souris. Cependant, leur capacité immunomodulatrice reste inconnue chez l’humain. Notre objectif consistait donc principalement à étudier le rôle et le mécanisme d’action des cellules T DN régulatrices humaines in vitro, en étudiant leur capacité à inhiber une réaction lymphocytaire mixte (MLR). Nous avons montré que les cellules T DN stimulées par un allo-antigène donné inhibent des cellules syngéniques effectrices dirigées contre ce même alloantigène mais n’inhibent pas des cellules syngéniques effectrices dirigées contre un autre alloantigène, démontrant ainsi la spécificité aux antigènes de ces cellules. De plus, les T DN non stimulées par un allo-antigène n’ont pas de rôle inhibiteur. Cependant, durant cette inhibition, nous n’observons pas de modulation de l’expression des marqueurs d’activation et d’induction de l’apoptose. Afin d’étudier le mécanisme d’action des cellules T DN, nous avons mesuré l’expression intracellulaire de la granzyme B. Les résultats démontrent que les cellules T DN stimulées expriment un niveau significativement plus élevé de granzyme B que les cellules T DN non-stimulées par l’allo-antigène. Ceci suggère que l’immunosuppression induite par les cellules T DN stimulées pourrait passer par la voie granzyme B. Le mécanisme utilisé par ces cellules reste à être confirmé par nos futures expériences. / Graft-versus-Host Disease (GvHD) is a major cause of morbidity and mortality in patients receiving an allogeneic Hematopoietic Stem Cell Transplantation (HSCT). Many regulatory T cell populations have been studied and shown to have immunosuppressive properties in GvHD. Among these populations, Double Negative CD4-CD8-TCRαβ+ regulatory T cells (DN T) have been described. These cells represent 1-3% of all T cell lymphocytes and are known to have antigen-specific inhibitory functions of the immune response. The suppression of an immune response by DN T cells seems to be an important mechanism involved in the induction of tolerance to allo-antigens. Moreover, these cells also confer immune tolerance in models of allogeneic and xenogenic grafts. DN T cells have the ability to suppress syngeneic T CD4+ and T CD8+ cells in an antigen-specific manner. Therefore, these DN T cells can inhibit the reaction caused by syngeneic effector cells against a specific alloantigen to which they have been previously exposed. However, they cannot inhibit a reaction directed against an unknown alloantigen. Human DN T cells have been isolated and characterized as cells that have the capacity to interact with APCs by cell-to-cell contact, just like in mice. However, their immunomodulatory properties are still unknown in humans. The goal of our project was to study the role and immunomodulatory functions of human DN T cells in Mixed Lymphocyte Reactions (MLR). The MLRs have allowed us to demonstrate that DN T cells, after having been stimulated by an allo-antigen, have an antigen-specific inhibitory function towards the syngeneic effector cells reacting against the same alloantigen that they have been exposed to. Interestingly, they do not inhibit the reaction of these effector cells against an unknown alloantigen. However, stimulated DN T cells did not modulate the expression of the activation markers expressed by the effector cells and did not give a death signal to these cells either. Moreover, we also wanted to study how DN T cells have an immunosuppressive activity. Therefore, we compared the expression of Granzyme B in stimulated versus non-stimulated cells. Our results suggest that DN T cells may use the Granzyme B pathway to immunosuppress the effector cells. In conclusion, our results demonstrate that DN T cells have an antigen-specific inhibitory function. The mechanism used by these DN T cells remains to be confirmed with our future experiments.

Survival of patients with poor prognosis or relapsed haematopoietic malignancies can be markedly improved by allogeneic haematopoietic stem cell transplantation (HSCT). HSCT reconstitutes the immune and haematopoietic systems after myeloablative conditioning and inhibits the recurrence of the malignancy by a graft-versus-leukaemia (GVL) response mediated by donor T cells. However, significant post-transplant complications such as graft-versus-host disease (GVHD) continue to plague the event-free survival of this curative procedure. GVHD is facilitated by donor T cells that recognise histocompatibility antigens on host antigen presenting cells (APC), such as dendritic cells (DC). Current treatment options for GVHD are focused on these T cells. However, these treatments result in an increased incidence of infection, graft rejection and relapse. A novel means of immunosuppression in GVHD is the use of multi-potent, mesenchymal stromal cells (MSC). MSC are non-immunogenic cells that actively suppress T cell function in vitro, and can resolve steroid-refractory GVHD in the clinic. Despite their use in the clinic, there is a paucity of pre-clinical data. Our aim was to investigate the in vivo efficacy of MSC to control GVHD while maintaining the beneficial GVL effect, and to begin to understand the mechanism by which MSC exert their immunosuppressive effects. We isolated and characterised MSC from murine bone/bone marrow and demonstrated that they suppressed T cell proliferation in vitro, even at low ratios of 1 MSC per 100 T cells. This was true of both donor-derived MSC, and MSC derived from unrelated donors (third party). Importantly, we observed that MSC significantly reduced T cell production of the pro-inflammatory cytokines TNFα and IFNγ in culture supernatants and that IFNγ plays a key role in the ability of MSC to suppress T cell proliferation. In vivo, we examined the effects of donor-derived MSC on GVHD severity and onset in two myeloablative murine models of HSCT. A major histocompatibility complex (MHC)-mismatched donor-recipient pair combination was used as a proof–of-principle model [UBI-GFP/BL6 (H-2b)àBALB/c (H-2d)], and an MHC-matched, minor histocompatibility antigen (miHA) mismatched donor-recipient pair combination was used to mimic MHC-matched sibling transplantation [UBI-GFP/BL6 (H-2b)àBALB.B (H-2b)]. We examined a number of variables related to MSC infusion including timing, dose and route of injection. We found that early post transplant infusion of MSC by the intraperitoneal injection was most effective at delaying death from GVHD, compared to pre-transplant infusion or intravenous injection. Furthermore, we found that the dose of MSC was critical, as infusion of too few MSC was ineffective and infusion of too many MSC exacerbated the development of GVHD. Taken together, these results suggest that timing, dose and route of injection are all important factors to be considered to ensure successful therapeutic outcome. To investigate the in vivo mechanism of action, we conducted timed sacrifice experiments in the MHC-mismatched model to determine if MSC altered cytokine secretion and cellular effectors, such as DC, known to play a key role in GVHD. Despite the fact that MSC given post-HSCT enter an environment full of activated DC and IFNγ levels, by day 3 and 6 post infusion, these activated DC and IFNγ levels are decreased compared to controls or mice infused with MSC pre-transplant (p<0.05). This confirmed our in vitro data that IFNγ played an important role in MSC-mediated immunosuppression. In addition, when we removed a major source of IFNγ production in vivo by administering the T cell depleting antibody KT3 to mice with or without MSC, we found that although T cell depletion prolonged survival, MSC were unable to further enhance this effect. This was also true when MSC were used in combination with the conventional immunosuppressant cyclosporine. Finally, we examined whether the infusion of MSC would compromise the GVL effect. We found that whilst MSC could delay the onset of GVHD, in our model they did not alter the anti-tumour effects of the donor T cells. Overall, we have shown that MSC can delay but not prevent death from GVHD when administered at an appropriate time and dose and that IFNγ is required for MSC-mediated immunosuppression in our model. These data suggest that patients undergoing HSCT should be monitored for IFNγ, and administered MSC when high levels are reached. Whilst MSC may be a promising therapy for patients with severe GVHD, we highlight that further investigation is warranted before MSC are accepted for widespread use in the clinic. The risks and benefits for transplant recipients should be carefully considered before utilising MSC to treat or prevent GVHD.

Survival of patients with poor prognosis or relapsed haematopoietic malignancies can be markedly improved by allogeneic haematopoietic stem cell transplantation (HSCT). HSCT reconstitutes the immune and haematopoietic systems after myeloablative conditioning and inhibits the recurrence of the malignancy by a graft-versus-leukaemia (GVL) response mediated by donor T cells. However, significant post-transplant complications such as graft-versus-host disease (GVHD) continue to plague the event-free survival of this curative procedure. GVHD is facilitated by donor T cells that recognise histocompatibility antigens on host antigen presenting cells (APC), such as dendritic cells (DC). Current treatment options for GVHD are focused on these T cells. However, these treatments result in an increased incidence of infection, graft rejection and relapse. A novel means of immunosuppression in GVHD is the use of multi-potent, mesenchymal stromal cells (MSC). MSC are non-immunogenic cells that actively suppress T cell function in vitro, and can resolve steroid-refractory GVHD in the clinic. Despite their use in the clinic, there is a paucity of pre-clinical data. Our aim was to investigate the in vivo efficacy of MSC to control GVHD while maintaining the beneficial GVL effect, and to begin to understand the mechanism by which MSC exert their immunosuppressive effects. We isolated and characterised MSC from murine bone/bone marrow and demonstrated that they suppressed T cell proliferation in vitro, even at low ratios of 1 MSC per 100 T cells. This was true of both donor-derived MSC, and MSC derived from unrelated donors (third party). Importantly, we observed that MSC significantly reduced T cell production of the pro-inflammatory cytokines TNFα and IFNγ in culture supernatants and that IFNγ plays a key role in the ability of MSC to suppress T cell proliferation. In vivo, we examined the effects of donor-derived MSC on GVHD severity and onset in two myeloablative murine models of HSCT. A major histocompatibility complex (MHC)-mismatched donor-recipient pair combination was used as a proof–of-principle model [UBI-GFP/BL6 (H-2b)àBALB/c (H-2d)], and an MHC-matched, minor histocompatibility antigen (miHA) mismatched donor-recipient pair combination was used to mimic MHC-matched sibling transplantation [UBI-GFP/BL6 (H-2b)àBALB.B (H-2b)]. We examined a number of variables related to MSC infusion including timing, dose and route of injection. We found that early post transplant infusion of MSC by the intraperitoneal injection was most effective at delaying death from GVHD, compared to pre-transplant infusion or intravenous injection. Furthermore, we found that the dose of MSC was critical, as infusion of too few MSC was ineffective and infusion of too many MSC exacerbated the development of GVHD. Taken together, these results suggest that timing, dose and route of injection are all important factors to be considered to ensure successful therapeutic outcome. To investigate the in vivo mechanism of action, we conducted timed sacrifice experiments in the MHC-mismatched model to determine if MSC altered cytokine secretion and cellular effectors, such as DC, known to play a key role in GVHD. Despite the fact that MSC given post-HSCT enter an environment full of activated DC and IFNγ levels, by day 3 and 6 post infusion, these activated DC and IFNγ levels are decreased compared to controls or mice infused with MSC pre-transplant (p<0.05). This confirmed our in vitro data that IFNγ played an important role in MSC-mediated immunosuppression. In addition, when we removed a major source of IFNγ production in vivo by administering the T cell depleting antibody KT3 to mice with or without MSC, we found that although T cell depletion prolonged survival, MSC were unable to further enhance this effect. This was also true when MSC were used in combination with the conventional immunosuppressant cyclosporine. Finally, we examined whether the infusion of MSC would compromise the GVL effect. We found that whilst MSC could delay the onset of GVHD, in our model they did not alter the anti-tumour effects of the donor T cells. Overall, we have shown that MSC can delay but not prevent death from GVHD when administered at an appropriate time and dose and that IFNγ is required for MSC-mediated immunosuppression in our model. These data suggest that patients undergoing HSCT should be monitored for IFNγ, and administered MSC when high levels are reached. Whilst MSC may be a promising therapy for patients with severe GVHD, we highlight that further investigation is warranted before MSC are accepted for widespread use in the clinic. The risks and benefits for transplant recipients should be carefully considered before utilising MSC to treat or prevent GVHD.