In this thesis, we report on various aspects of tumor - host (immune) interactions in Ewing sarcoma patients with the aim to obtain leads for immunotherapeutic or targeted treatment strategies. We... Show moreIn this thesis, we report on various aspects of tumor - host (immune) interactions in Ewing sarcoma patients with the aim to obtain leads for immunotherapeutic or targeted treatment strategies. We demonstrate a key role for interferon gamma (IFNg) in enhancing both Ewing sarcoma immunogenicity and susceptibility to cytotoxicity. An (endogenous) pro-inflammatory tumor microenvironment consisting of cytotoxic T-lymphocytes and IFNg-inducible chemokines provides prognostic benefit. Moreover, despite evidence for immunologic pressure in selection of HLA class I loss variants, intact IFNg-inducibility of the HLA class I antigen processing machinery emphasizes the significance of a pro-inflammatory microenvironment for initiation/ execution of adaptive anti-tumor immunity. Pre-clinical support for the potential of adoptive cell transfer therapies, in particular combinatorial natural killer cell-based therapy, is provide d. Sensitization of Ewing sarcoma by conventional or targeted therapies, including histone deacetylase inhibitors, combined with cytokine activation of natural killer cells enhances anti-tumor responses and overcomes both intrinsic functional natural killer cell defects as well as cross-resistance of chemotherapy-resistant Ewing sarcoma to natural killer cells. Finally, due to a crucial role of the CXCR4-CXCL12 axis in Ewing sarcoma progression, disruption of this axis by a CXCR4-specific antagonist may represent a promising treatment option for patients with Ewing sarcoma. Show less
The immune system plays an important role in the balance between viral clearance and viral persistence in HPV related (pre)malignant lesions. In this thesis, we analyzed HPV clade A9-specific T... Show moreThe immune system plays an important role in the balance between viral clearance and viral persistence in HPV related (pre)malignant lesions. In this thesis, we analyzed HPV clade A9-specific T-cell responses in relation to virological and clinical outcome to gain further insight into HPV-specific cellular immunity in relation to the natural course of disease. In depth analysis of cellular immune responses against the E6 antigen of HPV16 and the closely related members of clade A9 (HPV31, 33, 35, 52 and 58) showed us that HPV-specific cross-reactive CD4+ T cells are rare and unlikely to mediate cross protection (chapter 2). The clinical course of cervical HPV infection and HPV-specific immune responses in prospective studies are described in chapter 4 and 5. In those chapters, a strong correlation is observed between a persistent HPV infection or progressive disease and the lack or failure of a type-specific immune response (>90% of the cases). No correlation is detected between HPV type-specific cellular immune responses and virological clearance of the infection and HPV type-specific immunity may be associated with clearance of a cervical HPV induced lesion (chapter 4 and 5). Interestingly, a statistically significant trend could be detected between the presence of a type-specific immunity (HPV16E2) and regression of a low-grade lesion (chapter 5). A similar, but - due to small number of patients __ not statistically significant trend was observed in chapter 4. Together, this suggests that the local innate immune system might play a role in the clearance of transient infections, whereas the cellular immune response can play a role in regression of histologically proven HPV induced lesions. Detection of HPV16 type-specific cellular immune responses in vivo, by the use of a DTH skin test, confirmed that cellular immune responses in healthy subjects consist of both HPV-specific CD4+ Th1/Th2 and CD8+ T cells (chapter 3). Show less
This dissertation deals with the role of local immune stimulation in the lymph node and tumor microenvironment and its effect on systemic CD8+ T cell responses, in particular the anti-tumor CD8+ T... Show moreThis dissertation deals with the role of local immune stimulation in the lymph node and tumor microenvironment and its effect on systemic CD8+ T cell responses, in particular the anti-tumor CD8+ T cell responses. In chapter 2 the use of a slow-release system is described to deliver the immune-activating agonistic CD40 antibody to the tumor-draining area, and the advantages of this method over systemic administration of the antibody. The local, slow-release administration was very effective in activating a systemic anti-tumor effector CD8+ T cell response, to such an extent that a tenfold lower dose of antibody could be used without loss of efficacy. Adverse side-effects, analyzed by organ histology and liver enzymes in the blood, were much lower upon local anti-CD40 antibody delivery compared to systemic administration. The local delivery of anti-CD40 antibody resulted in a systemic anti-tumor CD8+ T cell response, capable of clearing distant tumors expressing identical tumor antigens. Chapter 3 shows that slow-release local administration of CTLA-4 blocking antibody can also activate a tumor-specific CD8+ T cell response and cause tumor regression, while lowering systemic adverse side-effect as compared to systemic administration. CTLA-4 blocking antibody is being widely used in clinical trials, and its use has been complicated by induction of auto-immune disease. Here we show that using a local low dose injection of CTLA-4 blocking antibody in a slow-release formulation is equally effective in activating a tumor-specific CD8+ T cell response, capable of eradicating tumor cells as systemic high dose treatment. The influence of local lymph node activation on systemic T cell responses is further analyzed in chapter 4. CD8+ T cell priming generally occurs in a locally inflamed lymph node, called a reactive LN, due to the presence of pathogens. The role of the inflammatory milieu on the priming and fate of CD8+ T cells was studied by separating the TCR-MHC interaction from the inflammatory cues, by priming briefly in vitro followed by transfer to mice with or without a CpG-induced reactive lymph node. The primary CD8+ T cell response was not influenced by the presence of a reactive lymph node, however, after a boost vaccination in the memory phase, CD8+ T cells primed in the presence of a reactive LN displayed a strong quantitative advantage over control CD8+ T cells. The reactive LN, which remained swollen with enhanced cellularity for a pronounced period of time, was envisaged to act as a shelter for CD8+ T cells while undergoing contraction after the primary response. In chapter 5, the advantages and disadvantages of the use of dextran-based microparticles as slow-release system for the delivery of immune-activating antibodies such as agonistic CD40 in the tumor-draining area are described. Dextran-based microparticles can be tailored to release antibodies in desired pharmacokinetics, leading to an even further decrease of adverse side-effects, as compared to previously described Montanide-ISA 51. However, dextran-based particles were unexpectedly found to have a stimulating effect on tumor-outgrowth. This effect coincided with the appearance of large, ulcerated swellings at the site of injection. In chapter 6, the issues presented in this thesis are discussed. The knowledge gained in the work shown here, compared with and strengthened by related published work, is used to state the opinion that targeting the tumor-draining lymph node and/or tumor microenvironment for immune-activating therapy against tumors must be seriously considered. Show less
For the successful application of immunotherapy for leukemia significant numbers of viable T cells with the right antigen specificity have to be available within a limited period of time. TCR gene... Show moreFor the successful application of immunotherapy for leukemia significant numbers of viable T cells with the right antigen specificity have to be available within a limited period of time. TCR gene transfer is a promising strategy enabling the generation of large numbers of T cells with a selective antigen specificity, while reducing the time required for in vitro culture. Future clinical application of TCR engineered T cells may result in an off-the-shelf therapy with TCRs suitable for large numbers of patients. Acquisition of more insight into the possibilities and restrictions of TCR gene transfer and assessment of the efficacy of the therapeutic cells may provide a solid basis for clinical application in the near future. However, the application of immunotherapy for hematological malignancies is currently limited by the restricted number of hematopoiesis specific mHags that can be used as target antigens. Furthermore, an important issue for clinical implementation of TCR gene therapy is the prevention of the formation of mixed TCR dimers, which have recently been shown to induce autoreactivity. In addition, the future development of TCR gene therapy strategies will benefit from increased knowledge about both the T cells that serve as host cell for TCR gene transfer and other T cells present in the patient that may manipulate the immunotherapeutic responses of the TCR engineered T cells. Unfortunately, function analysis of T cell populations using biologically relevant antigens is hampered by the large number of different T cell specificities present and the difficulty to identify the specificity of T cells. The aim of this thesis was to characterize novel T cell populations that may serve as host cells for TCR gene transfer and to determine their possibilities and restrictions as TCR engineered immunotherapeutic effector cells for the treatment of hematological malignancies. Show less
Type 1 diabetes is a common, multifactorial disease in which T cell autoimmunity plays an important role. Important research advances in recent years have fuelled the expectation that a definitive... Show moreType 1 diabetes is a common, multifactorial disease in which T cell autoimmunity plays an important role. Important research advances in recent years have fuelled the expectation that a definitive cure for the disease can be achieved for a large number of patients. In the first part of this thesis, the considerable clinical and immunological potential for intervention in the diabetogenic autoimmune process is described of the T cell costimulatory molecule CTLA4 and the autologous heat shock protein peptide DiaPep277. The second part focuses on islet and pancreas transplantation as a cure for type 1 diabetes. Next to the influence of allograft rejection and the effects of immunosuppressive therapy, associations are described between the presence and recurrence of diabetes-specific autoimmunity and clinical outcome of transplantation. Furthermore, possible factors are identified that may be modulated for improvement of outcome, and predictive biomarkers are suggested that may guide future treatment. In conclusion, this thesis recognizes T cell autoimmunity as an important entity in type 1 diabetes therapy, distinct from other types of immune reactivity. Tailored therapy for individual patients guided by close immune monitoring is being developed but needs to be improved further to establish a definitive cure for type 1 diabetes. Show less
Adoptive transfer of T cell immunity has been proposed as an attractive form of cancer immunotherapy in cases where the endogenous T cell repertoire is immune tolerant. In this thesis murine models... Show moreAdoptive transfer of T cell immunity has been proposed as an attractive form of cancer immunotherapy in cases where the endogenous T cell repertoire is immune tolerant. In this thesis murine models are used to study two different approaches to generate T cell grafts with defined tumor specificities. First the feasibility of minor antigen specific T cell depletion to limit the development of Graft-versus-Host Disease after allogeneic stem cell transplantation in combination with donor lymphocyte infusions is addressed. In the second part T cell receptor gene transfer is used to generate tumor specific T cells. Show less
Adoptive transfer of T-cells that are ex vivo selected for tumor-specificity is an attractive treatment strategy for cancer. Epstein Barr virus (EBV)-associated malignancies are ideal candidates to... Show moreAdoptive transfer of T-cells that are ex vivo selected for tumor-specificity is an attractive treatment strategy for cancer. Epstein Barr virus (EBV)-associated malignancies are ideal candidates to develop this type of immunotherapy as EBV-specific T-cells can readily be selected and expanded from peripheral blood of EBV-seropositive individuals. The first part of thesis describes a phase 1 clinical study which demonstrates the feasibility and safety of this approach in patients with advanced stage EBV-positive nasopharyngeal carcinoma. Unfortunately only a small number of cancers express viral antigens that are easily recognized by the immune system. Therefore a strategy is required to generate large numbers of T-cells specific for antigens that normally elicit no or only a weak immune response. In the second part of this thesis a method is described of engrafting T-cells with the required specificity using retroviral transfer of T-cell receptors (TCRs). The TCRs were further modified to incorporate costimulation signals (CD28, OX40) that are essential to initiate and sustain an effective anti-tumor response but are often lacking on the target tumor cells. Finally, an inducible safety switch was developed that allows for the ablation of the infused T-cells in vivo in case of toxicity, which will facilitate the implementation of these novel immunotherapy approaches in clinical studies. Show less
