En Pointe:Composing novel immunotherapy strategies to improve systemic anti-tumor immunity

Cancer is one of the leading causes of death and the global burden of cancer is predicted to increase to approximately 30 million new cases by 2040. The introduction of a new type of therapy, termed immunotherapy, has created a monumental breakthrough in cancer treatment. Despite being effective in a subset of patients with cancer, the majority of patients treated with immunotherapy do not experience durable clinical benefit. To increase the efficacy of immunotherapy, extensive research has focused on the tumor microenvironment (TME), but these efforts have not yet resulted in substantial improvements in therapeutic efficacy. The significance of coordinating regulation across various tissues is becoming apparent, highlighting the roles of the spleen, bone marrow, gut microbiome, and the tumor-draining lymph node (TDLN) in establishing systemic anti-tumor immunity. Understanding the dynamic coordination between cell types and their location in orchestrating anti-tumor immunity could provide novel insights in the requirements for an effective response to immunotherapy. Therefore, the aim of this thesis was to acquire in-depth understanding of mechanisms underlying the effectiveness and resistance of different immunotherapy strategies – immune checkpoint blockade (ICB) and dendritic cell (DC) therapy - by adopting a wider perspective of the systemic anti-tumor immune response. The results presented in this thesis highlight the importance of the tumor-draining lymph node (TDLN) in orchestrating anti-tumor immunity. More specifically, the TDLN appeared to be important in dictating the efficacy of anti-PD-1/PD-L1 immune checkpoint blockade. The importance of TDLN biology in establishing effective anti-tumor immunity was further underlined by findings showing that differences in TDLN immune contexture could underlie effective anti-tumor immunity as an immunosuppressive environment in the TDLN was related to disease recurrence in patients with melanoma. Besides a deeper understanding of the mechanisms of action, we also aimed to unravel novel mechanisms responsible for therapy resistance. To this end, we identified that anti-PD-1/PD-L1 immune checkpoint blockade could systemically activate regulatory T cells, causing therapy resistance in the process. Lastly, results presented in this thesis show that combining DC therapy with either anti-PD-1/PD-L1 immune checkpoint blockade or inhibition of janus kinase 3 could improve the efficacy of DC therapy. Together, the results presented in this thesis provide novel and important insights in the mode of action and mechanisms of resistance to immunotherapies, with a focus on anti-PD-1/PD-L1 immune checkpoint blockade and DC therapy. Importantly, these findings highlight that the effectiveness of immune responses directed at tumor cells is shaped by the intricate interactions among multiple tissues, rather than being exclusively dictated by conditions at the tumor site. These insights could offer novel avenues for improving existing strategies and could pave the way for the emergence of novel immunotherapies.<br/

En Pointe:Composing novel immunotherapy strategies to improve systemic anti-tumor immunity

Cancer is one of the leading causes of death and the global burden of cancer is predicted to increase to approximately 30 million new cases by 2040. The introduction of a new type of therapy, termed immunotherapy, has created a monumental breakthrough in cancer treatment. Despite being effective in a subset of patients with cancer, the majority of patients treated with immunotherapy do not experience durable clinical benefit. To increase the efficacy of immunotherapy, extensive research has focused on the tumor microenvironment (TME), but these efforts have not yet resulted in substantial improvements in therapeutic efficacy. The significance of coordinating regulation across various tissues is becoming apparent, highlighting the roles of the spleen, bone marrow, gut microbiome, and the tumor-draining lymph node (TDLN) in establishing systemic anti-tumor immunity. Understanding the dynamic coordination between cell types and their location in orchestrating anti-tumor immunity could provide novel insights in the requirements for an effective response to immunotherapy. Therefore, the aim of this thesis was to acquire in-depth understanding of mechanisms underlying the effectiveness and resistance of different immunotherapy strategies – immune checkpoint blockade (ICB) and dendritic cell (DC) therapy - by adopting a wider perspective of the systemic anti-tumor immune response. The results presented in this thesis highlight the importance of the tumor-draining lymph node (TDLN) in orchestrating anti-tumor immunity. More specifically, the TDLN appeared to be important in dictating the efficacy of anti-PD-1/PD-L1 immune checkpoint blockade. The importance of TDLN biology in establishing effective anti-tumor immunity was further underlined by findings showing that differences in TDLN immune contexture could underlie effective anti-tumor immunity as an immunosuppressive environment in the TDLN was related to disease recurrence in patients with melanoma. Besides a deeper understanding of the mechanisms of action, we also aimed to unravel novel mechanisms responsible for therapy resistance. To this end, we identified that anti-PD-1/PD-L1 immune checkpoint blockade could systemically activate regulatory T cells, causing therapy resistance in the process. Lastly, results presented in this thesis show that combining DC therapy with either anti-PD-1/PD-L1 immune checkpoint blockade or inhibition of janus kinase 3 could improve the efficacy of DC therapy. Together, the results presented in this thesis provide novel and important insights in the mode of action and mechanisms of resistance to immunotherapies, with a focus on anti-PD-1/PD-L1 immune checkpoint blockade and DC therapy. Importantly, these findings highlight that the effectiveness of immune responses directed at tumor cells is shaped by the intricate interactions among multiple tissues, rather than being exclusively dictated by conditions at the tumor site. These insights could offer novel avenues for improving existing strategies and could pave the way for the emergence of novel immunotherapies.<br/

Combination Strategies to Optimize Efficacy of Dendritic Cell-Based Immunotherapy

Dendritic cells (DCs) are antigen-presenting cells (APCs) that are essential for the activation of immune responses. In various malignancies, these immunostimulatory properties are exploited by DC-therapy, aiming at the induction of effective anti-tumor immunity by vaccination with ex vivo antigen-loaded DCs. Depending on the type of DC-therapy used, long-term clinical efficacy upon DC-therapy remains restricted to a proportion of patients, likely due to lack of immunogenicity of tumor cells, presence of a stromal compartment, and the suppressive tumor microenvironment (TME), thereby leading to the development of resistance. In order to circumvent tumor-induced suppressive mechanisms and unleash the full potential of DC-therapy, considerable efforts have been made to combine DC-therapy with chemotherapy, radiotherapy or with checkpoint inhibitors. These combination strategies could enhance tumor immunogenicity, stimulate endogenous DCs following immunogenic cell death, improve infiltration of cytotoxic T lymphocytes (CTLs) or specifically deplete immunosuppressive cells in the TME, such as regulatory T-cells and myeloid-derived suppressor cells. In this review, different strategies of combining DC-therapy with immunomodulatory treatments will be discussed. These strategies and insights will improve and guide DC-based combination immunotherapies with the aim of further improving patient prognosis and care

Combination Strategies to Optimize Efficacy of Dendritic Cell-Based Immunotherapy

Dendritic cells (DCs) are antigen-presenting cells (APCs) that are essential for the activation of immune responses. In various malignancies, these immunostimulatory properties are exploited by DC-therapy, aiming at the induction of effective anti-tumor immunity by vaccination with ex vivo antigen-loaded DCs. Depending on the type of DC-therapy used, long-term clinical efficacy upon DC-therapy remains restricted to a proportion of patients, likely due to lack of immunogenicity of tumor cells, presence of a stromal compartment, and the suppressive tumor microenvironment (TME), thereby leading to the development of resistance. In order to circumvent tumor-induced suppressive mechanisms and unleash the full potential of DC-therapy, considerable efforts have been made to combine DC-therapy with chemotherapy, radiotherapy or with checkpoint inhibitors. These combination strategies could enhance tumor immunogenicity, stimulate endogenous DCs following immunogenic cell death, improve infiltration of cytotoxic T lymphocytes (CTLs) or specifically deplete immunosuppressive cells in the TME, such as regulatory T-cells and myeloid-derived suppressor cells. In this review, different strategies of combining DC-therapy with immunomodulatory treatments will be discussed. These strategies and insights will improve and guide DC-based combination immunotherapies with the aim of further improving patient prognosis and care

Dendritic cell vaccination and CD40-agonist combination therapy licenses T cell-dependent antitumor immunity in a pancreatic carcinoma murine model

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is notoriously resistant to treatment including checkpoint-blockade immunotherapy. We hypothesized that a bimodal treatment approach consisting of dendritic cell (DC) vaccination to prime tumor-specific T cells, and a strategy to reprogram the desmoplastic tumor microenvironment (TME) would be needed to break tolerance to these pancreatic cancers. As a proof-of-concept, we investigated the efficacy of combined DC vaccination with CD40-agonistic antibodies in a poorly immunogenic murine model of PDAC. Based on the rationale that mesothelioma and pancreatic cancer share a number of tumor associated antigens, the DCs were loaded with either pancreatic or mesothelioma tumor lysates. METHODS: Immune-competent mice with subcutaneously or orthotopically growing KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) PDAC tumors were vaccinated with syngeneic bone marrow-derived DCs loaded with either pancreatic cancer (KPC) or mesothelioma (AE17) lysate and consequently treated with FGK45 (CD40 agonist). Tumor progression was monitored and immune responses in TME and lymphoid organs were analyzed using multicolor flow cytometry and NanoString analyzes. RESULTS: Mesothelioma-lysate loaded DCs generated cross-reactive tumor-antigen-specific T-cell responses to pancreatic cancer and induced delayed tumor outgrowth when provided as prophylactic vaccine. In established disease, combination with stimulating CD40 antibody was necessary to improve survival, while anti-CD40 alone was ineffective. Extensive analysis of the TME showed that anti-CD40 monotherapy did improve CD8 +T cell infiltration, but these essential effector cells displayed hallmarks of exhaustion, including PD-1, TIM-3 and NKG2A. Combination therapy induced a strong change in tumor transcriptome and mitigated the expression of inhibitory markers on CD8 +T cells. CONCLUSION: These results demonstrate the potency of DC therapy in combination with CD40-stimulation for the treatment of pancreatic cancer and provide directions for near future clinical trials

Combination of PD-1/PD-L1 checkpoint inhibition and dendritic cell therapy in mice models and in patients with mesothelioma

Immunotherapy with anti-PD1/PD-L1 is effective in only a subgroup of patients with malignant pleural mesothelioma (MPM). We investigated the efficacy of a combination of anti-PD1/PD-L1 and dendritic cell (DC) therapy to optimally induce effective anti-tumor immunity in MPM in both humans and mice. Data of nine MPM patients treated with DC therapy and sequential anti-PD1 treatment were collected and analyzed for progression-free survival (PFS) and overall survival (OS). Survival and T-cell responses were monitored in AC29 mesothelioma-bearing mice treated concurrently with the combination therapy; additionally, the role of the tumor-draining lymph node (TDLN) was investigated. The combination therapy resulted in a median OS and PFS of 17.7 and 8.0 months, respectively. Grade 3 to 4 treatment-related adverse events had not been reported. Survival of the mesothelioma-bearing mice treated with the combination therapy was longer than that of untreated mice, and coincided with improved T-cell activation in peripheral blood and less T-cell exhaustion in end stage tumors. Comparable results were obtained when solely the TDLN was targeted. We concluded that this combination therapy is safe and shows promising OS and PFS. The murine data support that PD-L1 treatment may reinvigorate the T-cell responses induced by DC therapy, which may primarily be the result of TDLN targeting

NKG2A is a late immune checkpoint on CD8 T cells and marks repeated stimulation and cell division

The surface inhibitory receptor NKG2A forms heterodimers with the invariant CD94 chain and is expressed on a subset of activated CD8 T cells. As antibodies to block NKG2A are currently tested in several efficacy trials for different tumor indications, it is important to characterize the NKG2A+ CD8 T cell population in the context of other inhibitory receptors. Here we used a well-controlled culture system to study the kinetics of inhibitory receptor expression. Naïve mouse CD8 T cells were synchronously and repeatedly activated by artificial antigen presenting cells in the presence of the homeostatic cytokine IL-7. The results revealed NKG2A as a late inhibitory receptor, expressed after repeated cognate antigen stimulations. In contrast, the expression of PD-1, TIGIT and LAG-3 was rapidly induced, hours after first contact and subsequently down regulated during each resting phase. This late, but stable expression kinetics of NKG2A was most similar to that of TIM-3 and CD39. Importantly, single-cell transcriptomics of human tumor-infiltrating lymphocytes (TILs) showed indeed that these receptors were often coexpressed by the same CD8 T cell cluster. Furthermore, NKG2A expression was associated with cell division and was promoted by TGF-β in vitro, although TGF-β signaling was not necessary in a mouse tumor model in vivo. In summary, our data show that PD-1 reflects recent TCR triggering, but that NKG2A is induced after repeated antigen stimulations and represents a late inhibitory receptor. Together with TIM-3 and CD39, NKG2A might thus mark actively dividing tumor-specific TILs

Immune monitoring in mesothelioma patients identifies novel immune-modulatory functions of gemcitabine associating with clinical response

Background: Gemcitabine is a frequently used chemotherapeutic agent but its effects on the immune system are incompletely understood. Recently, the randomized NVALT19-trial revealed that maintenance gemcitabine after first-line chemotherapy significantly prolonged progression-free survival (PFS) compared to best supportive care (BSC) in malignant mesothelioma. Whether these effects are paralleled by changes in circulating immune cell subsets is currently unknown. These analyses could offer improved mechanistic insights into the effects of gemcitabine on the host and guide development of effective combination therapies in mesothelioma. Methods: We stained peripheral blood mononuclear cells (PBMCs) and myeloid-derived suppressor cells (MDSCs) at baseline and 3 weeks following start of gemcitabine or BSC treatment in a subgroup of mesothelioma patients included in the NVALT19-trial. In total, 24 paired samples including both MDSCs and PBMCs were included. We performed multicolour flow-cytometry to assess co-inhibitory and-stimulatory receptor- and cytokine expression and matched these parameters with PFS and OS. Findings: Gemcitabine treatment was significantly associated with an increased NK-cell- and decreased T-regulatory cell proliferation whereas the opposite occurred in control patients. Furthermore, myeloid-derived suppressor cells (MDSCs) frequencies were lower in gemcitabine-treated patients and this correlated with increased T-cell proliferation following treatment. Whereas gemcitabine variably altered co-inhibitory receptor expression, co-stimulatory molecules including ICOS, CD28 and HLA-DR were uniformly increased across CD4+ T-helper, CD8+ T- and NK-cells. Although preliminary in nature, the increase in NK-cell proliferation and PD-1 expression in T cells following gemcitabine treatment was associated with improved PFS and OS. Interpretation: Gemcitabine treatment was associated with widespread effects on circulating immune cells of mesothelioma patients with responding patients displaying increased NK-cell and PD-1 + T-cell proliferation. These exploratory data provide a platform for future on treatment-biomarker development and novel combination treatment strategies

PD-L1 checkpoint blockade promotes regulatory T cell activity that underlies therapy resistance

Despite the clinical success of immune checkpoint blockade (ICB), in certain cancer types, most patients with cancer do not respond well. Furthermore, in patients for whom ICB is initially successful, this is often short-lived because of the development of resistance to ICB. The mechanisms underlying primary or secondary ICB resistance are incompletely understood. Here, we identified preferential activation and enhanced suppressive capacity of regulatory T cells (Treg cells) in αPD-L1 therapy-resistant solid tumor-bearing mice. Treg cell depletion reversed resistance to αPD-L1 with concomitant expansion of effector T cells. Moreover, we found that tumor-infiltrating Treg cells in human patients with skin cancer, and in patients with non-small cell lung cancer, up-regulated a suppressive transcriptional gene program after ICB treatment, which correlated with lack of treatment response. αPD-1/PD-L1-induced PD-1+ Treg cell activation was also seen in peripheral blood of patients with lung cancer and mesothelioma, especially in nonresponders. Together, these data reveal that treatment with αPD-1 and αPD-L1 unleashes the immunosuppressive role of Treg cells, resulting in therapy resistance, suggesting that Treg cell targeting is an important adjunct strategy to enhance therapeutic efficacy