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Nanovaccines, co-delivering antigen and invariant natural killer T (iNKT) cell agonists, proved to be very effective in inducing anti-tumor T cell responses due to their exceptional helper function. However, it is known that iNKT cells are not equally present in all lymphoid organs and nanoparticles do not get evenly distributed to all immune compartments. In this study, we evaluated the effect of the vaccination route on iNKT cell help to T and B cell responses for the first time in an antigen and agonist co-delivery setting. Intravenous administration of PLGA nanoparticles was mainly targeting liver and spleen where iNKT1 cells are abundant and induced the highest serum IFN-y levels, T cell cytotoxicity, and Th-1 type antibody responses. In comparison, after subcutaneous or intranodal injections, nanoparticles mostly drained or remained in regional lymph nodes where iNKT17 cells were abundant. After subcutaneous and intranodal injections, antigen-specific IgG2 c production was hampered and IFN-y production, as well as cytotoxic T cell responses, depended on sporadic systemic drainage. Therapeutic anti-tumor experiments also demonstrated a clear advantage of intravenous injection over intranodal or subcutaneous vaccinations. Moreover, tumor control could be further improved by PD-1 immune checkpoint blockade after intravenous vaccination, but not by intranodal vaccination. Anti PD-1 antibody combination mainly exerts its effect by prolonging the cytotoxicity of T cells. Nanovaccines also demonstrated synergism with anti-4-1BB agonistic antibody treatment in controlling tumor growth. We conclude that nanovaccines containing iNKT cell agonists shall be preferentially administered intravenously, to optimally reach cellular partners for inducing effective anti-tumor immune responses., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Chemotherapeutics, including the platinum compounds oxaliplatin (OXP) and cisplatin (CDDP), are standard care of treatment for cancer. Although chemotherapy has long been considered immunosuppressive, evidence now suggests that certain cytotoxic agents can efficiently stimulate antitumor responses, through the induction of a form of apoptosis, called immunogenic cell death (ICD). ICD is characterized by exposure of calreticulin and heat shock proteins (HSPs), secretion of ATP and release of high-mobility group box 1 (HMGB1). Proper activation of the immune system relies on the integration of these signals by dendritic cells (DCs). Studies on the crucial role of DCs, in the context of ICD, have been performed using mouse models or human in vitro-generated monocyte-derived DCs (moDCs), which do not fully recapitulate the in vivo situation. Here, we explore the effect of platinum-induced ICD on phenotype and function of human blood circulating DCs. Tumor cells were treated with OXP or CDDP and induction of ICD was investigated. We show that both platinum drugs triggered translocation of calreticulin and HSP70, as well as the release of ATP and HMGB1. Platinum treatment increased phagocytosis of tumor fragments by human blood DCs and enhanced phenotypic maturation of blood myeloid and plasmacytoid DCs. Moreover, upon interaction with platinum-treated tumor cells, CD1c(+) DCs efficiently stimulated allogeneic proliferation of T lymphocytes. Together, our observations indicate that platinum-treated tumor cells may exert an active stimulatory effect on human blood DCs. In particular, these data suggest that CD1c(+) DCs are critical mediators of immune responses induced by ICD.

Background: Ipilimumab has proven to be effective in metastatic melanoma patients. The purpose of this study was to determine the efficacy of ipilimumab in advanced melanoma patients who showed progressive disease upon experimental dendritic cell (DC) vaccination., Methods: Retrospective analysis of 48 stage IV melanoma patients treated with ipilimumab after progression upon DC vaccination earlier in their treatment. DC vaccination was given either as adjuvant treatment for stage III disease (n = 18) or for stage IV disease (n = 30). Ipilimumab (3 mg/kg) was administered every 3 weeks for up to 4 cycles., Results: Median time between progression upon DC vaccination and first gift of ipilimumab was 5.4 mo. Progression-free survival (PFS) rates for patients that received ipilimumab after adjuvant DC vaccination, and patients that received DC vaccination for stage IV melanoma, were 35% and 7% at 1 y and 35% and 3% at 2 y, while the median PFS was 2.9 mo and 3.1 mo, respectively. Median overall survival of patients pre-treated with adjuvant DC vaccination for stage III melanoma was not reached versus 8.0 mo (95% CI, 5.2-10.9) in the group pre-treated with DC vaccination for stage IV disease (HR of death, 0.36; p = 0.017). Grade 3 immune-related adverse events occurred in 19% of patients and one death (2%) was related to ipilimumab., Conclusions: Clinical responses to ipilimumab were found in a considerable number of advanced melanoma patients with progression after adjuvant DC vaccination for stage III disease, while the effect was very limited in patients who showed progression after DC vaccination for stage IV disease.

Purpose: To determine the effectiveness of adjuvant dendritic cell (DC) vaccination to induce tumor-specific immunological responses in stage III melanoma patients., Experimental Design: Retrospective analysis of stage III melanoma patients, vaccinated with autologous monocyte-derived DC loaded with tumor-associated antigens (TAA) gp100 and tyrosinase after radical lymph node dissection. Skin-test infiltrating lymphocytes (SKILs) obtained from delayed-type hypersensitivity skin-test biopsies were analyzed for the presence of TAA-specific CD8(+) T cells by tetrameric MHC-peptide complexes and by functional TAA-specific T cell assays, defined by peptide-recognition (T2 cells) and/or tumor-recognition (BLM and/or MEL624) with specific production of Th1 cytokines and no Th2 cytokines., Results: Ninety-seven patients were analyzed: 21 with stage IIIA, 34 with stage IIIB, and 42 had stage IIIC disease. Tetramer-positive CD8(+) T cells were present in 68 patients (70%), and 24 of them showed a response against all 3 epitopes tested (gp100:154-162, gp100:280-288, and tyrosinase:369-377) at any point during vaccinations. A functional T cell response was found in 62 patients (64%). Rates of peptide-recognition of gp100:154-162, gp100:280-288, and tyrosinase:369-377 were 40%, 29%, and 45%, respectively. Median recurrence-free survival and distant metastasis-free survival of the whole study population were 23.0 mo and 36.8 mo, respectively., Conclusions: DC vaccination induces a functional TAA-specific T cell response in the majority of stage III melanoma patients, indicating it is more effective in stage III than in stage IV melanoma patients. Furthermore, performing multiple cycles of vaccinations enhances the chance of a broader immune response.

Antitumor immunity can be enhanced by the coordinated release and delivery of antigens and immune-stimulating agents to antigen-presenting cells via biodegradable vaccine carriers. So far, encapsulation of TLR ligands and tumor-associated antigens augmented cytotoxic T cell (CTLs) responses. Here, we compared the efficacy of the invariant NKT (iNKT) cell agonist α-galactosylceramide (α-GalCer) and TLR ligands (R848 and poly I:C) as an adjuvant for the full length ovalbumin (OVA) in PLGA nanoparticles. We observed that OVA+α-GalCer nanoparticles (NP) are superior over OVA+TLR-L NP in generating and stimulating antigen-specific cytotoxic T lymphocytes without the need for CD4 + T cell help. Not only a 4-fold higher induction of antigen-specific T cells was observed, but also a more profound IFN-γ secretion was obtained by the addition α-GalCer. Surprisingly, we observed that mixtures of OVA containing NP with α-GalCer were ineffective, demonstrating that co-encapsulation of both α-GalCer and antigen within the same nanoparticle is essential for the observed T cell responses. Moreover, a single immunization with OVA+α-GalCer NP provided substantial protection from tumor formation and even delayed the growth of already established tumors, which coincided with a prominent and enhanced antigen-specific CD8 + T cell infiltration. The provided evidence on the advantage of antigen and α-GalCer coencapsulation should be considered in the design of future nanoparticle vaccines for therapeutic purposes.

Cytotoxic T cells are considered crucial for antitumor immunity and their induction is the aim of various immunotherapeutic strategies. High frequencies of tumor-specific CD8 + T cells alone, however, are no guarantee for long-term tumor control. Here, we analyzed the functionality of tumor-specific CD8 + T cells in melanoma patients upon dendritic cell vaccination by measuring multiple T cell effector functions considered crucial for anticancer immunity, including the expression of pro-inflammatory cytokines, chemokines and cytotoxic markers (IFNγ, TNFα, IL-2, CCL4, CD107a). We identified small numbers of multifunctional (polyfunctional) tumor-specific CD8 + T cells in several patients and dendritic cell therapy was able to improve the functionality of these pre-existing tumor-specific CD8 + T cells. Generated multifunctional CD8 + T cell responses could persist for up to ten years and within the same patient functionality could vary greatly for the different vaccination antigens. Importantly, after one cycle of DC vaccination highly functional CD8 + T cells were only detected in patients displaying prolonged overall survival. Our results shed light on the dynamics of multifunctional tumor-specific CD8 + T cells during metastatic melanoma and reveal a new feature of dendritic cell vaccination in vivo .

Melanoma patients with regional metastatic disease are at high risk for recurrence and metastatic disease, despite radical lymph node dissection (RLND). We investigated the immunologic response and clinical outcome to adjuvant dendritic cell (DC) vaccination in melanoma patients with regional metastatic disease who underwent RLND with curative intent. In this retrospective study, 78 melanoma patients with regional lymph node metastasis who underwent RLND received autologous DCs loaded with gp100 and tyrosinase and were analyzed for functional tumor-specific T cell responses in skin-test infiltrating lymphocytes. The study shows that adjuvant DC vaccination in melanoma patients with regional lymph node metastasis is safe and induced functional tumor-specific T cell responses in 71% of the patients. The presence of functional tumor-specific T cells was correlated with a better 2-year overall survival (OS) rate. OS was significantly higher after adjuvant DC vaccination compared to 209 matched controls who underwent RLND without adjuvant DC vaccination, 63.6 mo vs. 31.0 mo ( p = 0.018; hazard ratio 0.59; 95%CI 0.42-0.84). Five-year survival rate increased from 38% to 53% ( p < 0.01). In summary, in melanoma patients with regional metastatic disease, who are at high risk for recurrence and metastatic disease after RLND, adjuvant DC vaccination is well tolerated. It induced functional tumor-specific immune responses in the majority of patients and these were related to clinical outcome. OS was significantly higher compared to matched controls. A randomized clinical trial is needed to prospectively validate the efficacy of DC vaccination in the adjuvant setting.

Autologous dendritic cell (DC) therapy is an experimental cellular immunotherapy that is safe and immunogenic in patients with advanced melanoma. In an attempt to further improve the therapeutic responses, we treated 15 patients with melanoma, with autologous monocyte-derived immature DC electroporated with mRNA encoding CD40 ligand (CD40L), CD70 and a constitutively active TLR4 (caTLR4) together with mRNA encoding a tumor-associated antigen (TAA; respectively gp100 or tyrosinase). In addition, DC were pulsed with keyhole limpet hemocyanin (KLH) that served as a control antigen. Production of this DC vaccine with high cellular viability, high expression of co-stimulatory molecules and MHC class I and II and production of IL-12p70, was feasible in all patients. A vaccination cycle consisting of three vaccinations with up to 15×10 6 DC per vaccination at a biweekly interval, was repeated after 6 and 12 months in the absence of disease progression. mRNA-optimized DC were injected intranodally, because of low CCR7 expression on the DC, and induced de novo immune responses against control antigen. T cell responses against tyrosinase were detected in the skin-test infiltrating lymphocytes (SKIL) of two patients. One mixed tumor response and two durable tumor stabilizations were observed among 8 patients with evaluable disease at baseline. In conclusion, autologous mRNA-optimized DC can be safely administered intranodally to patients with metastatic melanoma but showed limited immunological responses against tyrosinase and gp100.

Bioassays that predict clinical outcome are essential to optimize cellular anticancer immunotherapy. We have recently developed a robust and simple skin test to evaluate the capacity of tumor-specific T cells to migrate, recognize their targets and exert effector functions. This bioassay detects T cells with an elevated antineoplastic potential and hence rapidly identifies patients responding to immunotherapy.

Dendritic cell (DC)-based vaccines require the cells to relocate to lymph nodes (LNs). Unfortunately, however, DC migration rates are typically very poor. We investigated strategies to increase the migration efficacy of DC-based vaccines. Surprisingly, a reduction in DC number, but not the conditioning of the injection site, improved LN targeting.

Dendritic cell-based anticancer immunotherapy is feasible, safe and results in the induction of tumor-specific immune responses, at least in a fraction of vaccinated patients. The concomitant activation of cytotoxic and helper T cells, by loading DCs with peptides or electroporating them with the corresponding mRNAs, may further enhance vaccine-induced antitumor responses.

The identification of growth and differentiation pathways that are responsible for the proliferation and survival of cancer stem cells (CSCs) has opened avenues for the discovery of novel therapeutic targets. In the initial phase of an anticancer immune response, T cells specific for tumor-associated antigens develop in patients and, at least under selected circumstances, are able to eliminate malignant cells. However, it remains unknown whether CSC-specific T cells are also operational. We found naturally occurring multifunctional CD4 + and CD8 + T cells specific for the stem cell marker OCT4 among the peripheral blood mononuclear cells (PBMCs) of both healthy individuals and ovarian cancer patients. Moreover, lymphocytes isolated from the ascites of patients affected by ovarian malignancies also contained OCT4-specific T cells. OCT4-reactive CD4 + T cells did not produce interferon γ (IFNγ) and IFNγ-inducible protein 10 (IP-10) but were capable of proliferation upon stimulation with dendritic cells (DCs) loaded with an OCT4-derived peptide or OCT4 mRNA. OCT4-reactive CD8 + cells did not proliferate in response to a similar challenge, yet produced IP-10 as well as sufficient amounts of IFNγ to induce IP-10 . Furthermore, CD8 + cytotoxic T cells were able to release their lysosomal components, as indicated by the mobilization of CD107a. These results demonstrate the existence of anti-CSC specific T cells in ovarian cancer patients.

Dendritic cell-based immunotherapy is a promising strategy against cancer that appears to be feasible, safe and to induce potent tumor-specific immune responses. The use of naturally circulating dendritic cells (DCs), rather than cultured monocyte-derived DCs, might constitute the next logical step to translate anticancer immune responses into long-lasting clinical benefits.

In order for cellular therapeutics to succeed, comprehensive monitoring of the transplanted cells in vivo is required i.e., their localization, functionality and numbers in a longitudinal manner. Recently, dendritic cell based vaccines have been monitored by their effect on lymphocyte activation using [(18)F]FLT PET in cancer patients.

Background: The success of dendritic cell (DC)-based immunotherapy in inducing cellular immunity against tumors is highly dependent on accurate delivery and trafficking of the DC to T-cell-rich areas of secondary lymphoid tissues., Objective: To provide an overview of DC migration in vivo and how migration to peripheral lymph nodes might be improved to optimize DC therapy., Methods: We focused on DC migration in preclinical models and human skin explants and on clinical vaccination trials studying migration of in vitro-generated DC., Results/conclusions: DC migration requires an intricate interplay between the cell and its environment. To maximize migration for cellular therapy, it is important to optimize the generation of migratory DC as well as treatment strategies.

Dendritic cells are the most potent antigen-presenting cells of the immune system and represent a promising tool in therapeutic vaccination against cancer. Immunotherapy applying ex vivo-generated and tumor antigen-loaded dentritic cells has been successfully introduced in clinical vaccination protocols and has proven to be feasible and effective in some patients. A better understanding of how dentritic cells succeed to induce and modulate immunity is necessary to optimally exploit dentritic cells in anticancer vaccines. The authors will review novel insights in antigen loading, activation and migration of dentritic cells and their impact on the application of ex vivo-generated dentritic cell vaccines. In addition, novel means to exploit dentritic cells in cancer vaccines by loading and activation of dentritic cells directly in situ and possible obstacles that should be overcome to induce long-lasting immunity in therapeutic settings will be discussed.

Leukocyte function associated antigen 1 (LFA-1) is an adhesion molecule indispensable in immune and inflammatory reactions, but its role in hematopoiesis is poorly understood. LFA-1 is considered as a marker of late stage stem cell maturation when expressed on CD34(+) bone marrow cells. We observed that CD34(+) bone marrow cells express LFA-1, that based on LFA-1 expression several subpopulations can be distinguished, and that the level of expression appeared highly variable among different donors. Unanticipated, in time course experiments we observed that CD34(+) LFA-1(-) cells expressed LFA-1 within 24 hours upon culture. These in vitro findings support the hypothesis that once contacts with bone marrow stroma are lost, LFA-1 is upregulated by default, due to the lack of negative regulating signals from stromal cells. This might also explain the widely variable expression of LFA-1 as a result of crowding of cells in the bone marrow with subsequent loss of contact with stroma and upregulation of LFA-1, thus equipping cells with adequate adhesion receptors to migrate throughout the bone marrow. Interestingly, the expression of the LFA-1 specific activation epitope L16 on these cells is low, even after culture. This demonstrated that LFA-1 is not activated, as was confirmed by low adhesion to ICAM-1. Activation of adhesion molecules is induced by several growth factors. Indeed, we show here that an osteoblastic cell line under normal conditions does hardly produce hematopoietic growth factors but these are rapidly induced after stimulation. Such rapid induction endows the bone marrow stroma with the property to modulate the adhesive strength and enabling migration through the different environments within the stroma. Prove for such compartments within the bone marrow is provided by histological data.