In vitro expansion of Wilms' tumor protein 1 epitope-specific primary T cells from healthy human peripheral blood mononuclear cells.

Wilms' tumor protein 1 (WT1) is a tumor-associated antigen overexpressed in various cancers. As a self-antigen, negative selection reduces the number of WT1-specific T cell receptors (TCRs). Here, we provide a protocol to generate WT137-45-specific TCRs using healthy human peripheral blood mononuclear cells. We describe the expansion of WT1-specific T cell clones by two consecutive in vitro stimulations with autologous WT137-45-pulsed dendritic cells and peripheral blood lymphocytes. We then detail the detection with human leukocyte antigen/WT137-45 tetramers.

Anti-Tumor Potency of Short-Term Interleukin-15 Dendritic Cells Is Potentiated by In Situ Silencing of Programmed-Death Ligands.

Dendritic cell (DC) vaccines have proven to be a valuable tool in cancer immune therapy. With several DC vaccines being currently tested in clinical trials, knowledge about their therapeutic value has been significantly increased in the past decade. Despite their established safety, it has become clear that objective clinical responses are not yet robust enough, requiring further optimization. Improvements of this advanced therapy medicinal product encompass, among others, regulating their immune stimulating capacity by in situ gene engineering, in addition to their implementation in combination therapy regimens. Previously, we have reported on a superior monocyte-derived DC preparation, including interleukin-15, pro-inflammatory cytokines and immunological danger signals in the culture process. These so-called IL-15 DCs have already proven to exhibit several favorable properties as cancer vaccine. Evolving research into mechanisms that could further modulate the immune response towards cancer, points to programmed death-1 as an important player that dampens anti-tumor immunity. Aiming at leveraging the immunogenicity of DC vaccines, we hypothesized that additional implementation of the inhibitory immune checkpoint molecules programmed death-ligand (PD-L)1 and PD-L2 in IL-15 DC vaccines would exhibit superior stimulatory potential. In this paper, we successfully implemented PD-L silencing at the monocyte stage in the 3-day IL-15 DC culture protocol resulting in substantial downregulation of both PD-L1 and PD-L2 to levels below 30%. Additionally, we validated that these DCs retain their specific characteristics, both at the level of phenotype and interferon gamma secretion. Evaluating their functional characteristics, we demonstrate that PD-L silencing does not affect the capacity to induce allogeneic proliferation. Ultimately designed to induce a durable tumor antigen-specific immune response, PD-L silenced IL-15 DCs were capable of surpassing PD-1-mediated inhibition by antigen-specific T cells. Further corroborating the superior potency of short-term IL-15 DCs, the combination of immune stimulatory components during DC differentiation and maturation with in situ checkpoint inhibition supports further clinical translation.

Serum profiling identifies ibrutinib as a treatment option for young adults with B-cell acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) is a haematological malignancy that is characterized by the uncontrolled proliferation of immature lymphocytes. 80% of cases occur in children where ALL is well understood and treated. However it has a devastating affects on adults, where multi-agent chemotherapy is the standard of care with allogeneic stem cell transplantation for those who are eligible. New treatments are required to extend remission and prevent relapse to improve patient survival rates. We used serum profiling to compare samples from presentation adult B-ALL patients with age- and sex-matched healthy volunteer (HV) sera and identified 69 differentially recognised antigens (P ≤ 0·02). BMX, DCTPP1 and VGLL4 showed no differences in transcription between patients and healthy donors but were each found to be present at higher levels in B-ALL patient samples than HVs by ICC. BMX plays a crucial role in the Bruton's Tyrosine Kinase (BTK) pathway which is bound by the BTK inhibitor, ibrutinib, suggesting adult B-ALL would also be a worthy target patient group for future clinical trials. We have shown the utility of proto-array analysis of B-ALL patient sera, predominantly from young adults, to help characterise the B-ALL immunome and identified a new target patient population for existing small molecule therapy.

New targets for therapy: antigen identification in adults with B-cell acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) in adults is a rare and difficult-to-treat cancer that is characterised by excess lymphoblasts in the bone marrow. Although many patients achieve remission with chemotherapy, relapse rates are high and the associated impact on survival devastating. Most patients receive chemotherapy and for those whose overall fitness supports it, the most effective treatment to date is allogeneic stem cell transplant that can improve overall survival rates in part due to a 'graft-versus-leukaemia' effect. However, due to the rarity of this disease, and the availability of mature B-cell antigens on the cell surface, few new cancer antigens have been identified in adult B-ALL that could act as targets to remove residual disease in first remission or provide alternative targets for escape variants if and when current immunotherapy strategies fail. We have used RT-PCR analysis, literature searches, antibody-specific profiling and gene expression microarray analysis to identify and prioritise antigens as novel targets for the treatment of adult B-ALL.

Dendritic Cell-Based and Other Vaccination Strategies for Pediatric Cancer.

Dendritic cell-based and other vaccination strategies that use the patient's own immune system for the treatment of cancer are gaining momentum. Most studies of therapeutic cancer vaccination have been performed in adults. However, since cancer is one of the leading causes of death among children past infancy in the Western world, the hope is that this form of active specific immunotherapy can play an important role in the pediatric population as well. Since children have more vigorous and adaptable immune systems than adults, therapeutic cancer vaccines are expected to have a better chance of creating protective immunity and preventing cancer recurrence in pediatric patients. Moreover, in contrast to conventional cancer treatments such as chemotherapy, therapeutic cancer vaccines are designed to specifically target tumor cells and not healthy cells or tissues. This reduces the likelihood of side effects, which is an important asset in this vulnerable patient population. In this review, we present an overview of the different therapeutic cancer vaccines that have been studied in the pediatric population, with a main focus on dendritic cell-based strategies. In addition, new approaches that are currently being investigated in clinical trials are discussed to provide guidance for further improvement and optimization of pediatric cancer vaccines.

Chimeric Antigen Receptor-Modified T Cell Therapy in Multiple Myeloma: Beyond B Cell Maturation Antigen.

Chimeric antigen receptor (CAR)-modified T cell therapy is a rapidly emerging immunotherapeutic approach that is revolutionizing cancer treatment. The impressive clinical results obtained with CAR-T cell therapy in patients with acute lymphoblastic leukemia and lymphoma have fueled the development of CAR-T cells targeting other malignancies, including multiple myeloma (MM). The field of CAR-T cell therapy for MM is still in its infancy, but remains promising. To date, most studies have been performed with B cell maturation antigen (BCMA)-targeted CARs, for which high response rates have been obtained in early-phase clinical trials. However, responses are usually temporary, and relapses have frequently been observed. One of the major reasons for relapse is the loss or downregulation of BCMA expression following CAR-T therapy. This has fostered a search for alternative target antigens that are expressed on the MM cell surface. In this review, we provide an overview of myeloma target antigens other than BCMA that are currently being evaluated in pre-clinical and clinical studies.

CD56 Homodimerization and Participation in Anti-Tumor Immune Effector Cell Functioning: A Role for Interleukin-15.

A particularly interesting marker to identify anti-tumor immune cells is the neural cell adhesion molecule (NCAM), also known as cluster of differentiation (CD)56. Namely, hematopoietic expression of CD56 seems to be confined to powerful effector immune cells. Here, we sought to elucidate its role on various killer immune cells. First, we identified the high motility NCAM-120 molecule to be the main isoform expressed by immune cells. Next, through neutralization of surface CD56, we were able to (1) demonstrate the direct involvement of CD56 in tumor cell lysis exerted by CD56-expressing killer cells, such as natural killer cells, gamma delta (γδ) T cells, and interleukin (IL)-15-cultured dendritic cells (DCs), and (2) reveal a putative crosstalk mechanism between IL-15 DCs and CD8 T cells, suggesting CD56 as a co-stimulatory molecule in their cell-to-cell contact. Moreover, by means of a proximity ligation assay, we visualized the CD56 homophilic interaction among cancer cells and between immune cells and cancer cells. Finally, by blocking the mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3K)-Akt pathway, we showed that IL-15 stimulation directly led to CD56 upregulation. In conclusion, these results underscore the previously neglected importance of CD56 expression on immune cells, benefiting current and future immune therapeutic options.

Dendritic Cell-Based Immunotherapy of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a type of blood cancer characterized by the uncontrolled clonal proliferation of myeloid hematopoietic progenitor cells in the bone marrow. The outcome of AML is poor, with five-year overall survival rates of less than 10% for the predominant group of patients older than 65 years. One of the main reasons for this poor outcome is that the majority of AML patients will relapse, even after they have attained complete remission by chemotherapy. Chemotherapy, supplemented with allogeneic hematopoietic stem cell transplantation in patients at high risk of relapse, is still the cornerstone of current AML treatment. Both therapies are, however, associated with significant morbidity and mortality. These observations illustrate the need for more effective and less toxic treatment options, especially in elderly AML and have fostered the development of novel immune-based strategies to treat AML. One of these strategies involves the use of a special type of immune cells, the dendritic cells (DCs). As central orchestrators of the immune system, DCs are key to the induction of anti-leukemia immunity. In this review, we provide an update of the clinical experience that has been obtained so far with this form of immunotherapy in patients with AML.

Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing.

Genetic engineering of T cells with tumor specific T-cell receptors (TCR) is a promising strategy to redirect their specificity against cancer cells in adoptive T cell therapy protocols. Most studies are exploiting integrating retro- or lentiviral vectors to permanently introduce the therapeutic TCR, which can pose serious safety issues when treatment-related toxicities would occur. Therefore, we developed a versatile, non-genotoxic transfection method for human unstimulated CD8+ T cells. We describe an optimized double sequential electroporation platform whereby Dicer-substrate small interfering RNAs (DsiRNA) are first introduced to suppress endogenous TCR α and ß expression, followed by electroporation with DsiRNA-resistant tumor-specific TCR mRNA. We demonstrate that double sequential electroporation of human primary unstimulated T cells with DsiRNA and TCR mRNA leads to unprecedented levels of transgene TCR expression due to a strongly reduced degree of TCR mispairing. Importantly, superior transgenic TCR expression boosts epitope-specific CD8+ T cell activation and killing activity. Altogether, DsiRNA and TCR mRNA double sequential electroporation is a rapid, non-integrating and highly efficient approach with an enhanced biosafety profile to engineer T cells with antigen-specific TCRs for use in early phase clinical trials.

BDCA1+CD14+ Immunosuppressive Cells in Cancer, a Potential Target?

Dendritic cell (DC) vaccines show promising effects in cancer immunotherapy. However, their efficacy is affected by a number of factors, including (1) the quality of the DC vaccine and (2) tumor immune evasion. The recently characterized BDCA1+CD14+ immunosuppressive cells combine both aspects; their presence in DC vaccines may directly hamper vaccine efficacy, whereas, in patients, BDCA1+CD14+ cells may suppress the induced immune response in an antigen-specific manner systemically and at the tumor site. We hypothesize that BDCA1+CD14+ cells are present in a broad spectrum of cancers and demand further investigation to reveal treatment opportunities and/or improvement for DC vaccines. In this review, we summarize the findings on BDCA1+CD14+ cells in solid cancers. In addition, we evaluate the presence of BDCA1+CD14+ cells in leukemic cancers. Preliminary results suggest that the presence of BDCA1+CD14+ cells correlates with clinical features of acute and chronic myeloid leukemia. Future research focusing on the differentiation from monocytes towards BDCA1+CD14+ cells could reveal more about their cell biology and clinical significance. Targeting these cells in cancer patients may improve the outcome of cancer immunotherapy.

A versatile T cell-based assay to assess therapeutic antigen-specific PD-1-targeted approaches.

Blockade of programmed cell death protein 1 (PD-1) immune checkpoint receptor signaling is an established standard treatment for many types of cancer and indications are expanding. Successful clinical trials using monoclonal antibodies targeting PD-1 signaling have boosted preclinical research, encouraging development of novel therapeutics. Standardized assays to evaluate their bioactivity, however, remain restricted. The robust bioassays available all lack antigen-specificity. Here, we developed an antigen-specific, short-term and high-throughput T cell assay with versatile readout possibilities. A genetically modified T cell receptor (TCR)-deficient T cell line was stably transduced with PD-1. Transfection with messenger RNA encoding a TCR of interest and subsequent overnight stimulation with antigen-presenting cells, results in eGFP-positive and granzyme B-producing T cells for single cell or bulk analysis. Control antigen-presenting cells induced reproducible high antigen-specific eGFP and granzyme B expression. Upon PD-1 interaction, ligand-positive antigen-presenting immune or tumor cells elicited significantly lower eGFP and granzyme B expression, which could be restored by anti-PD-(L)1 blocking antibodies. This convenient cell-based assay shows a valuable tool for translational and clinical research on antigen-specific checkpoint-targeted therapy approaches.

The role of the common gamma-chain family cytokines in γδ T cell-based anti-cancer immunotherapy.

Cytokines of the common gamma-chain receptor family, comprising interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21, are vital with respect to organizing and sustaining healthy immune cell functions. Supporting the anti-cancer immune response, these cytokines inspire great interest for their use as vaccine adjuvants and cancer immunotherapies. It is against this background that gamma delta (γδ) T cells, as special-force soldiers and natural contributors of the tumor immunosurveillance, also received a lot of attention the last decade. As γδ T cell-based cancer trials are coming of age, this present review focusses on the effects of the different cytokines of the common gamma-chain receptor family on γδ T cells with respect to boosting γδ T cells as a therapeutic target in cancer immunotherapy. This review also gathers data that IL-15 in particular exhibits key features for augmenting the anti-tumor activity of effector killer γδ T cells whilst overcoming the myriad of immune escape mechanisms used by cancer cells.

Interleukin-15-Cultured Dendritic Cells Enhance Anti-Tumor Gamma Delta T Cell Functions through IL-15 Secretion.

Dendritic cell (DC) vaccination can be an effective post-remission therapy for acute myeloid leukemia (AML). Yet, current DC vaccines do not encompass the ideal stimulatory triggers for innate gamma delta (γδ) T cell anti-tumor activity. Promoting type 1 cytotoxic γδ T cells in patients with AML is, however, most interesting, considering these unconventional T cells are primed for rapid function and exert meaningful control over AML. In this work, we demonstrate that interleukin (IL)-15 DCs have the capacity to enhance the anti-tumoral functions of γδ T cells. IL-15 DCs of healthy donors and of AML patients in remission induce the upregulation of cytotoxicity-associated and co-stimulatory molecules on the γδ T cell surface, but not of co-inhibitory molecules, incite γδ T cell proliferation and stimulate their interferon-γ production in the presence of blood cancer cells and phosphoantigens. Moreover, the innate cytotoxic capacity of γδ T cells is significantly enhanced upon interaction with IL-15 DCs, both towards leukemic cell lines and allogeneic primary AML blasts. Finally, we address soluble IL-15 secreted by IL-15 DCs as the main mechanism behind the IL-15 DC-mediated γδ T cell activation. These results indicate that the application of IL-15-secreting DC subsets could render DC-based anti-cancer vaccines more effective through, among others, the involvement of γδ T cells in the anti-leukemic immune response.

Dendritic Cells and Programmed Death-1 Blockade: A Joint Venture to Combat Cancer.

Two decades of clinical cancer research with dendritic cell (DC)-based vaccination have proved that this type of personalized medicine is safe and has the capacity to improve survival, but monotherapy is unlikely to cure the cancer. Designed to empower the patient's antitumor immunity, huge research efforts are set to improve the efficacy of next-generation DC vaccines and to find synergistic combinations with existing cancer therapies. Immune checkpoint approaches, aiming to breach immune suppression and evasion to reinforce antitumor immunity, have been a revelation in the immunotherapy field. Early success of therapeutic antibodies blocking the programmed death-1 (PD-1) pathway has sparked the development of novel inhibitors and combination therapies. Hence, merging immunoregulatory tumor-specific DC strategies with PD-1-targeted approaches is a promising path to explore. In this review, we focus on the role of PD-1-signaling in DC-mediated antitumor immunity. In the quest of exploiting the full potential of DC therapy, different strategies to leverage DC immunopotency by impeding PD-1-mediated immune regulation are discussed, including the most advanced research on targeted therapeutic antibodies, lessons learned from chemotherapy-induced immune activation, and more recent developments with soluble molecules and gene-silencing techniques. An overview of DC/PD-1 immunotherapy combinations that are currently under preclinical and clinical investigation substantiates the clinical potential of such combination strategies.

On the feasibility of mining CD8+ T cell receptor patterns underlying immunogenic peptide recognition.

Current T cell epitope prediction tools are a valuable resource in designing targeted immunogenicity experiments. They typically focus on, and are able to, accurately predict peptide binding and presentation by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. However, recognition of the peptide-MHC complex by a T cell receptor (TCR) is often not included in these tools. We developed a classification approach based on random forest classifiers to predict recognition of a peptide by a T cell receptor and discover patterns that contribute to recognition. We considered two approaches to solve this problem: (1) distinguishing between two sets of TCRs that each bind to a known peptide and (2) retrieving TCRs that bind to a given peptide from a large pool of TCRs. Evaluation of the models on two HIV-1, B*08-restricted epitopes reveals good performance and hints towards structural CDR3 features that can determine peptide immunogenicity. These results are of particular importance as they show that prediction of T cell epitope and T cell epitope recognition based on sequence data is a feasible approach. In addition, the validity of our models not only serves as a proof of concept for the prediction of immunogenic T cell epitopes but also paves the way for more general and high-performing models.

Increased herpes zoster risk associated with poor HLA-A immediate early 62 protein (IE62) affinity.

Around 30% of individuals will develop herpes zoster (HZ), caused by the varicella zoster virus (VZV), during their life. While several risk factors for HZ, such as immunosuppressive therapy, are well known, the genetic and molecular components that determine the risk of otherwise healthy individuals to develop HZ are still poorly understood. We created a computational model for the Human Leukocyte Antigen (HLA-A, -B, and -C) presentation capacity of peptides derived from the VZV Immediate Early 62 (IE62) protein. This model could then be applied to a HZ cohort with known HLA molecules. We found that HLA-A molecules with poor VZV IE62 presentation capabilities were more common in a cohort of 50 individuals with a history of HZ compared to a nationwide control group, which equated to a HZ risk increase of 60%. This tendency was most pronounced for cases of HZ at a young age, where other risk factors are less prevalent. These findings provide new molecular insights into the development of HZ and reveal a genetic predisposition in those individuals most at risk to develop HZ.

Dendritic cell vaccination as postremission treatment to prevent or delay relapse in acute myeloid leukemia.

Relapse is a major problem in acute myeloid leukemia (AML) and adversely affects survival. In this phase 2 study, we investigated the effect of vaccination with dendritic cells (DCs) electroporated with Wilms' tumor 1 (WT1) messenger RNA (mRNA) as postremission treatment in 30 patients with AML at very high risk of relapse. There was a demonstrable antileukemic response in 13 patients. Nine patients achieved molecular remission as demonstrated by normalization of WT1 transcript levels, 5 of which were sustained after a median follow-up of 109.4 months. Disease stabilization was achieved in 4 other patients. Five-year overall survival (OS) was higher in responders than in nonresponders (53.8% vs 25.0%; P = .01). In patients receiving DCs in first complete remission (CR1), there was a vaccine-induced relapse reduction rate of 25%, and 5-year relapse-free survival was higher in responders than in nonresponders (50% vs 7.7%; P < .0001). In patients age ≤65 and >65 years who received DCs in CR1, 5-year OS was 69.2% and 30.8% respectively, as compared with 51.7% and 18% in the Swedish Acute Leukemia Registry. Long-term clinical response was correlated with increased circulating frequencies of polyepitope WT1-specific CD8+ T cells. Long-term OS was correlated with interferon-γ+ and tumor necrosis factor-α+ WT1-specific responses in delayed-type hypersensitivity-infiltrating CD8+ T lymphocytes. In conclusion, vaccination of patients with AML with WT1 mRNA-electroporated DCs can be an effective strategy to prevent or delay relapse after standard chemotherapy, translating into improved OS rates, which are correlated with the induction of WT1-specific CD8+ T-cell response. This trial was registered at www.clinicaltrials.gov as #NCT00965224.

CD56 in the Immune System: More Than a Marker for Cytotoxicity?

Over the past years, the phenotypic and functional boundaries distinguishing the main cell subsets of the immune system have become increasingly blurred. In this respect, CD56 (also known as neural cell adhesion molecule) is a very good example. CD56 is the archetypal phenotypic marker of natural killer cells but can actually be expressed by many more immune cells, including alpha beta T cells, gamma delta T cells, dendritic cells, and monocytes. Common to all these CD56-expressing cell types are strong immunostimulatory effector functions, including T helper 1 cytokine production and an efficient cytotoxic capacity. Interestingly, both numerical and functional deficiencies and phenotypic alterations of the CD56+ immune cell fraction have been reported in patients with various infectious, autoimmune, or malignant diseases. In this review, we will discuss our current knowledge on the expression and function of CD56 in the hematopoietic system, both in health and disease.

Monocyte-Derived Dendritic Cells with Silenced PD-1 Ligands and Transpresenting Interleukin-15 Stimulate Strong Tumor-Reactive T-cell Expansion.

Although allogeneic stem cell transplantation (allo-SCT) can elicit graft-versus-tumor (GVT) immunity, patients often relapse due to residual tumor cells. As essential orchestrators of the immune system, vaccination with dendritic cells (DC) is an appealing strategy to boost the GVT response. Nevertheless, durable clinical responses after DC vaccination are still limited, stressing the need to improve current DC vaccines. Aiming to empower DC potency, we engineered monocyte-derived DCs to deprive them of ligands for the immune checkpoint regulated by programmed death 1 (PD-1). We also equipped them with interleukin (IL)-15 "transpresentation" skills. Transfection with short interfering (si)RNA targeting the PD-1 ligands PD-L1 and PD-L2, in combination with IL15 and IL15Rα mRNA, preserved their mature DC profile and rendered the DCs superior in inducing T-cell proliferation and IFNγ and TNFα production. Translated into an ex vivo hematological disease setting, DCs deprived of PD-1 ligands (PD-L), equipped with IL15/IL15Rα expression, or most effectively, both, induced superior expansion of minor histocompatibility antigen-specific CD8+ T cells from transplanted cancer patients. These data support the combinatorial approach of in situ suppression of the PD-L inhibitory checkpoints with DC-mediated IL15 transpresentation to promote antigen-specific T-cell responses and, ultimately, contribute to GVT immunity. Cancer Immunol Res; 5(8); 710-5. ©2017 AACR.

Desirable cytolytic immune effector cell recruitment by interleukin-15 dendritic cells.

Success of dendritic cell (DC) therapy in treating malignancies is depending on the DC capacity to attract immune effector cells, considering their reciprocal crosstalk is partially regulated by cell-contact-dependent mechanisms. Although critical for therapeutic efficacy, immune cell recruitment is a largely overlooked aspect regarding optimization of DC vaccination. In this paper we have made a head-to-head comparison of interleukin (IL)-15-cultured DCs and conventional IL-4-cultured DCs with regard to their proficiency in the recruitment of (innate) immune effector cells. Here, we demonstrate that IL-4 DCs are suboptimal in attracting effector lymphocytes, while IL15 DCs provide a favorable chemokine milieu for recruiting CD8+ T cells, natural killer (NK) cells and gamma delta (γδ) T cells. Gene expression analysis revealed that IL-15 DCs exhibit a high expression of chemokines involved in antitumor immune effector cell attraction, while IL-4 DCs display a more immunoregulatory profile characterized by the expression of Th2 and regulatory T cell-attracting chemokines. This is confirmed by functional data indicating an enhanced recruitment of granzyme B+ effector lymphocytes by IL-15 DCs, as compared to IL-4 DCs, and subsequent superior killing of tumor cells by the migrated lymphocytes. Elevated CCL4 gene expression in IL-15 DCs and lowered CCR5 expression on both migrated γδ T cells and NK cells, led to validation of increased CCL4 secretion by IL15 DCs. Moreover, neutralization of CCR5 prior to migration resulted in an important inhibition of γδ T cell and NK cell recruitment by IL-15 DCs. These findings further underscore the strong immunotherapeutic potential of IL-15 DCs.