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Neoadjuvant ipilimumab plus nivolumab showed high pathologic response rates (pRRs) in patients with macroscopic stage III melanoma in the phase 1b OpACIN () and phase 2 OpACIN-neo () studies(1,2). While the results are promising, data on the durability of these pathologic responses and baseline biomarkers for response and survival were lacking. After a median follow-up of 4 years, none of the patients with a pathologic response (n = 7/9 patients) in the OpACIN study had relapsed. In OpACIN-neo (n = 86), the 2-year estimated relapse-free survival was 84% for all patients, 97% for patients achieving a pathologic response and 36% for nonresponders (P < 0.001). High tumor mutational burden (TMB) and high interferon-gamma-related gene expression signature score (IFN-gamma score) were associated with pathologic response and low risk of relapse; pRR was 100% in patients with high IFN-gamma score/high TMB; patients with high IFN-gamma score/low TMB or low IFN-gamma score/high TMB had pRRs of 91% and 88%; while patients with low IFN-gamma score/low TMB had a pRR of only 39%. These data demonstrate long-term benefit in patients with a pathologic response and show the predictive potential of TMB and IFN-gamma score. Our findings provide a strong rationale for a randomized phase 3 study comparing neoadjuvant ipilimumab plus nivolumab versus standard adjuvant therapy with antibodies against the programmed cell death protein-1 (anti-PD-1) in macroscopic stage III melanoma.

Adoptive T cell therapy (ATT) can achieve regression of large tumors in mice and humans; however, tumors frequently recur. High target peptide-major histocompatibility complex-I (pMHC) affinity and T cell receptor (TCR)-pMHC affinity are thought to be critical to preventing relapse. Here, we show that targeting two epitopes of the same antigen in the same cancer cells via monospecific T cells, which have similar pMHC and pMHC-TCR affinity, results in eradication of large, established tumors when targeting the apparently subdominant but not the dominant epitope. Only the escape but not the rejection epitope required postproteasomal trimming, which was regulated by IFN-gamma, allowing IFN-gamma-unresponsive cancer variants to evade. The data describe a novel immune escape mechanism and better define suitable target epitopes for ATT.

T-cell recognition of minor histocompatibility antigens (MiHA) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, the number of MiHA identified to date remains limited, making clinical application of MiHA reactive T-cell infusion difficult. This study represents the first attempt of genome-wide prediction of MiHA, coupled to the isolation of T-cell populations that react with these antigens. In this unbiased high-throughput MiHA screen, both the possibilities and pitfalls of this approach were investigated. First, 973 polymorphic peptides expressed by hematopoietic stem cells were predicted and screened for HLA-A2 binding. Subsequently a set of 333 high affinity HLA-A2 ligands was identified and post transplantation samples from allo-SCT patients were screened for T-cell reactivity by a combination of pMHC-tetramer-based enrichment and multi-color flow cytometry. Using this approach, 71 peptide-reactive T-cell populations were generated. The isolation of a T-cell line specifically recognizing target cells expressing the MAP4K1(IMA) antigen demonstrates that identification of MiHA through this approach is in principle feasible. However, with the exception of the known MiHA HMHA1, none of the other T-cell populations that were generated demonstrated recognition of endogenously MiHA expressing target cells, even though recognition of peptide-loaded targets was often apparent. Collectively these results demonstrate the technical feasibility of high-throughput analysis of antigen-specific T-cell responses in small patient samples. However, the high-sensitivity of this approach requires the use of potential epitope sets that are not solely based on MHC binding, to prevent the frequent detection of T-cell responses that lack biological relevance.

Resident memory CD8+ T cells (CD8+ TRM) play a central role in tissue immunity. This thesis focusses on the formation, function and behavior of CD8+ TRM in the skin. Firstly, by making use of lineage tracing technology, we show that the capacity to form TRM is instilled in CD8+ T cells before the cells enter the affected tissue and that this capacity remains fixed after a secondary immune challenge. Second, this works shows that CD8+ skin-TRM act as local sentinels that trigger a rapid and tissue-wide immune response upon foreign antigen recognition. Third, ex vivo imaging of human skin biopsies demonstrates that tissue patrol is a property of CD8+ TRM in human skin. In addition, this work describes the development of two novel technologies (i.e. the ‘Tol’ mouse strain and the ex vivo imaging technique) that can be used as such or adapted by other biomedical researchers in order to investigate key aspects of (T) cell behavior and skin biology. Understanding the signals that drive CD8+ TRM formation, and insights in the function of these cells within both healthy and diseased skin, will be essential to allow the manipulation of these populations for therapeutic benefit.

Tumor organoids are 3D cultures of cancer cells. Research into the applications of tumor organoids as a model system for cancer has received great interest. Here, we critically evaluate which opportunities this model systems offers, and which challenges still need to be overcome. In chapter 1, we describe the resemblance of organoids and the tumors they are derived from, and culture success rates for organoids of various tumor types. Organoids are compared to cell lines and patient-derived xenografts. We offer a perspective on potential applications for individualized medicine. In chapter 2, we make the connection with immunotherapy. Although the efficacy of immunotherapy is high, many patients do not respond to treatment. We discuss how genetic analyses can be used to identify those patients that benefit most from treatment. In chapter 3, we generate and characterize organoids of 3 different neuroendocrine carcinomas (NEC). Few preclinical models exist for this rare and understudied tumor type. These patients were treated with everolimus and cisplatin, and clinical response matches drug response of organoids. This chapter shows the potential of organoids to model rare tumors. In chapters 4 and 5, organoid models are expanded by the inclusion of immune cells. T cells from peripheral blood of cancer patients are co-cultured with tumor organoids from the same patient. This leads to tumor recognition by T cells in a subset of patients. These T cells specifically attack tumor organoids, but ignore healthy colon or lung organoids. This offers the potential to model the interaction between immune cells and cancer cells at the level of the individual patient. In chapter 6, we critically evaluate lung cancer organoids. Establishment of organoids from intrapulmonary tumors is complicated by the growth of normal airway organoids. We describe that this often leads to contamination of cultures by normal airway organoids. It is essential to determine the purity of tumor organoid cultures. We systematically describe the suitability of various methods to do so. The eventual culture success of pure lung cancer organoids is 17%, making it difficult to use this for personalized medicine. In chapter 6 we determine the suitability of organoids to predict a tumor’s sensitivity to chemotherapy. We find that sensitivity of colon cancer to irinotecan-based therapies can be predicted very well. In contrast, there is no predictive value for oxaliplatin-based therapies. This indicates that the use of tumor organoids In chapter 8 we define the role of our developed T cell – organoid co-culture system in the context of a clinical study. We study in detail a patient with two synchronous tumors, that differ in response to immunotherapy. We see that autologous T cells only recognize organoids from the sensitive tumor. We do not see differences between the two tumors when T cells specific to a model antigen are used. We describe the value and possible limitations of these different experimental setups. We conclude that organoid – T cell co-culture systems can be useful if the research question takes into account the limitations of a reductionist model system.

This thesis focused on different aspects of melanoma treatment with immunotherapy and targeted therapy. In chapter 2 we search for biomarkers that could be associated with overall survival in patients treated with ipilimumab. In chapter 3 we describe diarrea, a commonly seen side effect of immunotherapy. Here we show that there is no significant correlation between grade of diarrhea and severity of colitis as seen during endoscopy. Chapters 4 and 5 describe patients with brain metastases and/or leptomeningeal metastases. In chapter 4 we show the difference in overall survival in patients treated with vemurafenib, dabrafenib or the combination of dabrafenib + trametinib. Chapter 5 focusses on the treatment of leptomeningeal metastases. Here a significant difference in overall survival was noted between treated and untreated patients. Furthermore LDH was a predictive biomarker for overall survival. In chapter 6 we show that treating patients with vemurafenib beyond progression of disease has a significant impact on overall survival. Lastly in chapter 7 we review the past, present and future of treating patients with different kinds of cancer with tumor-infiltrating lymphocytes.

Infusions of T cells engineered with TCRs directed against tumor-expressed antigens have demonstrated clinical efficacy in the treatment of cancer. To broaden the applicability of this approach a broad repertoire of clinically relevant TCRs is required. This thesis describes a high throughput methodology for the isolation of TCRs useful in the treatment of patients suffering from B-cell malignancies. To induce strong immune responses against tumor-expressed antigens, the immunogencity of allogeneic (non-self) HLA molecules is exploited. Around this fundamental knowledge about T-cell reactivity a pipeline is developed that consists of the selection of targetable tumor-expressed antigens by mining gene expression databases, discovery of HLA-presented peptides from peptide elution studies describing the HLA ligandome of B lymphocytes, a peptide-MHC tetramer based high throughput methodology for the isolation and characterization of T cells expressing TCRs specific for tumor-expressed antigens. Several TCRs were isolated targeting antigens that are expressed on several B-cell malignancies including multiple myeloma. This is of particular interest since novel immunotherapies for multiple myeloma are currently lacking. The work described in this thesis can significantly broaden the applicability of immunotherapy by providing novel tools and reagents for strategies relying on tumor recognition through antigen presentation in the context of HLA.

Cancer immunotherapy has taken up its place in oncological practice and it is likely there to stay. Although the field has come from far, much is still to learn as oncologists observe that not all patients with the same disease type respond to cancer immunotherapy, Furthermore, in some cancer types, no clinical benefit is observed at all, despite the presence of an abundant local immune infiltrate, indicating that our understanding of cancer – immune interaction is still incomplete. The overall goal of this thesis is to help address these matters in three ways. First, we have retrospectively analyzed a cohort of melanoma patients treated with ipilimumab, an anti-CTLA-4 antibody, for markers of responsiveness and review the literature to explore the limitations and curative potential of treatment strategies currently used in the clinic or under development. Second, we have developed several technology platforms that allow us to characterize and compare different types of T-cell responses that are directed against tumor-specific antigens in patient tumor material. Third, we aim to increase our understanding of the potential of cancer immunotherapy in ovarian and colorectal cancer, by analysis of the tumor-reactive compartment within these diseases.

The subject of this thesis is the study of the function and behavior of CD8+ T cells that have exited the circulation and entered a tissue in order to get rid of an infection. In particular, the research focus on 1- how effector CD8+ T cells can localize infected cells within a tissue. 2-how, once the infection has been cleared, effector CD8+ T cells become memory T cells that take up permanent residence in the tissue; also, how the patrolling behavior that these cells acquire in the tissue allows quick control of local secondary infections. 3-how tissue-resident memory CD8+ T cells that have re-encountered a specific antigen can induce an increased level of local immune protection against infections.

T cell responses against tumor-antigens are frequently observed for some human malignancies, in particular melanoma. However, the spontaneous development of T cell responses of a sufficient strength to eradicate human malignancies is rare. The transfer of T cell receptor (TCR) __ genes into autologous T cells, a process called TCR gene transfer, allows one to engineer antigen-specific T cell responses of interest. Over the last decade, TCR gene transfer has progressed to the stage of clinical evaluation as a strategy to engineer T cell responses against tumor-antigens, in order to treat metastatic melanoma and other malignancies. The scope of the work in this thesis is to advance the development of TCR gene transfer as an engineering strategy for T cell immunity against cancer. This involves the assessment of the safety of TCR gene transfer, the evaluation of strategies to enhance the function of TCR-modified T cells in vivo, and the development of technologies to identify tumor-reactive TCR__ genes

Melanoma is a malignancy that arises from melanocytes, the pigment-producing cells that can be predominantly found in the eye or the epidermal basal layer of the skin. Mainly due to increased UV exposure, the incidence of melanoma has doubled worldwide over the past three decades (200.000 new cases in 2008). Primary melanomas can be easily treated by surgical resection, leading to a good prognosis for stage I patients. However, metastasized melanoma is almost completely resistant to therapeutic modalities such as radio- and chemotherapy, resulting in a median overall survival of less than one year for this patient group. Despite considerable efforts, for over 20 years there was no melanoma treatment developed that could improve survival of stage IV patients. However, the treatment of unresectable metastasized melanoma has progressed markedly in recent years due to the development of both immunotherapies that stimulate anti-tumor immunity and targeted therapies that block oncogenic proteins. This thesis will focus on pre-clinical work concerning the optimization of melanoma treatment. In detail, it will address for both targeted therapies and immunotherapies factors that play a role in the identification of response-predictive biomarkers, the toxicity of treatments, and the potential efficacy of combination treatments.

This thesis describes the development and use of a novel technology for single-cell fate mapping, called cellular barcoding. With this technology, unique and heritable genetic tags (barcodes) are introduced into na_ve T cells. Using cellular barcoding, we investigated I) how different antigen-specific CD8+ T cell clones contribute to the formation of effector and memory T cell subsets II) at what point during in vivo CD8+ T cell differentiation fate decisions take place and III) to what extent the clonal expansion of individual antigen-specific CD8+ T cells shapes the overall response magnitude. Our experiments demonstrate that most if not all effector and memory T cells are progeny of the same na_ve T cells and that the decision to develop into either subset is taken after the first cell division. Furthermore, we show that the overall T cell response magnitude is primarily controlled by the extent of clonal expansion, as the efficiency by which na_ve T cells are recruited into the response is remarkably constants. Quantification of barcode abundances revealed that individual antigen-specific T cells produce highly variable numbers of daughter cells, and that this strong disparity in output is established during the first phase of infection.

Following antigen encounter, activated T cells can give rise to functionally distinct T cell subsets. Understanding how different T cell subsets arise requires technologies that can monitor the developmental potential of single precursor cells (chapter 2). This thesis describes the development and use of two novel genetic tagging strategies aimed at following cell differentiation in vivo. These strategies are based on the marking of precursor cells with unique DNA sequences (barcodes), following which cell fate is analyzed by barcode comparison of different daughter populations. The first technology, termed cellular barcoding, makes use of a retroviral barcode library to provide T cells with unique genetic tags via in vitro transduction. Cellular barcoding was used to analyze the kinship of diverse T cell populations (chapter 3-5) as well as to measure the clonality of antigen-specific T cell responses under varying conditions of infection (chapter 6). The second technology, termed in vivo barcoding, makes use of a transgenic mouse model in which unique DNA sequences are introduced via inducible VDJ recombination. The feasibility of in vivo barcoding was demonstrated by conditionally labeling lung and liver cells with different barcodes (chapter 7). Together, these studies have yielded important new insights for vaccine optimization.

This thesis focuses on the generation of MHC ligands and their use in analyzing T cell immunity, both in mouse and men. It is roughly split into two sections: the first part deals specifically with the rules governing the generation of MHC ligands, while the second part describes technological advances in the use of these MHC ligands to analyze T cell immunity. The first part of this thesis starts with an introduction on antigen presentation, covering both the different mechanisms through which epitopes are generated and the process by which epitopes are presented to T cells. Emphasis lies on the discoveries in this field of the last decade. This introduction is followed by two chapters on the generation of antigenic epitopes. Chapter two describes the analysis of protease activity in the endocytic pathway, while chapter three investigates the antigenic source of epitopes generated during cross-presentation. The second part of this thesis starts with a review on the status of MHC multimer technology as a tool to analyze antigen specific T cell populations. Chapter five then describes a high-throughput approach to generate a vast array of different peptide-MHC complexes for several human MHC alleles, allowing for faster and more complex applications of MHC multimer technology. In the next chapter, the peptide-exchange technology of chapter 5 is used for the development of another novel strategy: multidimensional encoding of peptide-MHC complexes. This chapter describes the development, validation and use of this encoding technique that allows the parallel detection of antigen-specific T cell responses of up to 25 T cell populations in one single sample. Finally, this thesis concludes with a summary and discussion chapter, giving a short overview of the presented data and discussing its relevance in the field of antigen presentation.

Cancer cells often escape the attack of immune cells because they originate from self-tissue. Through T cell receptor gene transfer it is possible to equip peripheral T cells with a desired specificity, and this strategy may be useful to generate tumor-specific T cells for the treatment of cancer in humans. This thesis describes several aspects in the functionality, safety and longevity of T cells modified by T cell receptor gene transfer in animal models.

This thesis describes different immunotherapeutic strategies that can be used for the treatment of cancer in general, and of melanoma in particular. Tumor-specific T cell responses can be induced via either active or passive immunization. Active immunization can be used to target tumors for which high affinity T cells are present within the endogenous repertoire. This thesis describes a vaccination method where a tattoo device is used to deliver a DNA vaccine into the skin. The vaccine-induced anti-tumor effect can be further enhanced by combining vaccination with host conditioning and adoptive transfer. Passive immunization via TCR gene transfer is the preferred strategy when targeting tumor self antigens, because this can circumvent limitations of the endogenous T cell repertoire and does not require extensive in vitro culture. This thesis describes several factors that can improve the anti-tumor ac tivity of TCR modified cells. In addition, we describe a procedure for the selection of a human TCR that is well expressed and highly affine, and that is therefore likely to enable more efficient targeting of human melanoma. Implementation of these factors in the design of clinical trials is likely to positively affect the clinical efficacy of TCR gene therapy.

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 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.

T cells recognize pathogen-derived antigens and are crucial for fighting pathogens such as viruses and bacteria. In addition, T cells are able to recognize and attack certain types of tumors, in particular virally induced tumors. In this thesis we aimed 1) to obtain more insight into antigen-specific T cell responses and 2) to study how antigen-specific T cell responses can be improved. For the first aim we generated new tools that by enabling the visualization of antigen-specific CD4+ and CD8+ T cells allow the study of the dynamics of antigen-specific T cell responses in time throughout an ongoing immune response (chapter 2). In addition, we developed a novel technique that enables the study of family relationships between different T cell populations. This technique for instance allows us to determine whether two different types of effector T cell populations arise from the same or different pool(s) of na_ve T cells (chapter 5). For the second aim, we analyzed whether antigen-specific T cell responses can be manipulated by providing increased costimulation in the form of constitutive triggering of CD27 (chapter 3) or by generating CD4+ T cells that are modified by the introduction of MHC class I restricted TCRs (chapter 4).