Novel immunotherapy for high-risk neuroblastoma: allogeneic stem cell transplantation and recipient leukocyte infusion.

Neuroblastoma is the most frequent extra-cranial solid tumor in childhood. More than half of patients is diagnosed with high-risk disease (non-resectable and metastasized), and is faced with poor prognosis. To date, an effective and safe treatment for such patients is not available, urgently necessitating research into novel treatment strategies. Allogeneic hematopoietic stem cell transplantation is a cornerstone in the treatment of hematological malignancies, particularly in combination with donor leukocyte infusion (DLI), and is now also emerging as a novel treatment option for advanced solid tumors not responding to conventional therapies. The major drawback is that donor T cells not only attack malignant cells (graft-versus-tumor effect), but also target healthy recipient tissues resulting in graft-versus-host disease (GvHD), causing high morbidity and mortality. Studies in murine leukemia models have shown that infusion of recipient-type – rather than donor-type – leukocytes (recipient leukocyte infusion, RLI) after allogeneic stem cell transplantation can also mount anti-tumor responses without producing GvHD: RLI given to mixed chimeras provoked an immune reaction against the allogeneic cells, resulting in a full rejection of the donor graft with associated anti-leukemia effects, while leaving host tissues intact. Importantly, we were the first group to show that RLI can also produce immune responses against the solid tumor neuroblastoma: in murine neuroblastoma models we showed that RLI is able to reduce local growth and metastasis of neuroblastoma, which was initiated by a complete rejection of donor T-cell chimerism. However, although local tumor growth was significantly delayed by RLI, the effect was temporarily and survival was not prolonged. Therefore, the aim of the current project was to gain a detailed insight into the nature and the dynamics of the alloreactive response following RLI, so as to understand why the biological effects are incomplete, and to explore strategies to optimize the efficacy of RLI against neuroblastoma. In the current project, we revealed that the RLI-mediated alloreactive response was associated with a systemic inflammatory cytokine storm, expansion of activated dentritic cells (DC) in the lymph nodes and induction of a marked systemic expansion of host-type T helper 1 cells and cytotoxic T cells that were also able to migrate into the local neuroblastoma tumor. However, although the systemic effector T cell responses persisted for -at least- 2 weeks after RLI, within the tumor the effect diminished more rapidly. Accordingly, we found that RLI not only generated effector T cell responses, but also drives a strong expansion of myeloid-derived suppressor cells (MDSC). Depletion of MDSC by low-dose 5-fluorouracil augmented the anti-tumor effects of RLI, indicating that the expanded MDSC are - at least in part - responsible for counteracting the tumor growth-inhibitory effect of RLI.Table of contents DANKWOORD LIST OF ABBREVIATIONS III CHAPTER 1 1 INTRODUCTION 1 1. NEUROBLASTOMA. 1 1.1 Epidemiology and pathogenesis. 1 1.2 Diagnosis, staging and treatment stratification. 2 1.3 Conventional treatment of high-risk neuroblastoma. 5 1.4 Immunosurveillance, cancer immunoediting and neuroblastoma immunogenicity. 7 2. ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION. 9 2.1 The graft-versus-leukemia effect. 10 2.1.1 Curative potential of alloHSCT. 10 2.1.2 Mechanisms of the GvL effect and association with GvHD. 11 Donor T lymphocytes. 11 Donor NK cells. 14 2.2 Donor leukocyte infusion to enhance the GvL effect of alloHSCT. 16 2.3 The graft-versus-solid tumor effect. 19 3. RECIPIENT IMMUNE CELLS IN THE GRAFT-VERSUS-TUMOR EFFECT: IT TAKES TWO TO TANGO 23 3.1 Contribution of recipient cells to anti-tumor effects. 23 3.2 Recipient leukocyte infusion for the induction of anti-tumor effects. 25 3.2.1 RLI to enhance anti-leukemia effects. 26 3.2.2 RLI to enhance the anti-neuroblastoma effect. 29 CHAPTER 2 35 AIM OF THE STUDY 35 CHAPTER 3 37 MATERIALS AND METHODS 37 CHAPTER 4 45 RESULTS 45 Part I: The cellular dynamics of RLI-mediated lymphohematopoietic alloreactivity, and induction of effector cell pathways 45 1. DYNAMICS OF THE RLI-INDUCED LYMPHOHEMATOPOIETIC ALLOREACTIVITY AND THE TUMOR GROWTH-INHIBITORY EFFECT. 48  1.1 RLI delays local neuroblastoma tumor growth and mediates a strong lymphohematopoietic host-versus-graft reaction. 48 1.2 RLI mediates a systemic expansion of host T cells and a decrease in total B and NK cell number 49 1.3 RLI alloreactivity is associated with production of inflammatory cytokines. 53 2. INDUCTION OF EFFECTOR T CELL PATHWAYS BY RLI. 55 2.1 RLI induces an expansion of activated DC in the lymph nodes. 55 2.2 RLI generates host-type Th1 and CTL responses in lymph nodes and spleen. 57 2.3 RLI allows infiltration of effector T cells into the tumor. 61 2.3.1 Neuroblastoma cells up-regulate MHC class I after RLI. 61 2.3.2 Tumors of RLI chimeras show accumulation of host-type Th1 cells and CTL. 65 Part II: Potential immunoregulatory pathways and strategies to improve the efficacy of RLI 71 1. REGULATORY T CELLS AND MYELOID-DERIVED SUPPRESSOR CELLS AFTER RLI. 74 1.1 Regulatory T cells are not increased after RLI. 74 1.2 RLI is followed by a marked expansion of myeloid-derived suppressor cells. 76 1.3 RLI-associated expanding MDSC are suppressors of chimeric T cells. 80 1.4 Immunosuppressive mechanisms of RLI-induced MDSC. 82 2. TARGETING MDSC TO IMPROVE THE ANTI-TUMOR EFFECT OF RLI. 85 2.1 Low-dose 5-fluorouracil selectively depletes MDSC in vivo. 85 2.2 5-FU-mediated MDSC depletion enhances the tumor growth-inhibitory effect of RLI. 89 2.3 Low-dose 5-FU is not cytotoxic for Neuro2A. 91 CHAPTER 5 97 GENERAL DISCUSSION AND FUTURE PERSPECTIVES 97 CHAPTER 6 109 SUMMARY 109 CHAPTER 7 113 SAMENVATTING 113 CHAPTER 8 117 ACKNOWLEDGEMENTS, PERSONAL CONTRIBUTION AND CONFLICT OF INTEREST STATEMENTS 117 REFERENCES 121 CURRICULUM VITAE 139 BIBLIOGRAPHY 141nrpages: 162status: accepte