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Targeting dendritic cells (DC) through the release of suppressive factors is an effective means for tumors to escape immune control. We assessed the involvement of downstream signaling through the JAK2/STAT3 and p38 MAPK pathways in tumor-induced suppression of human DC development. Whereas the JAK2/STAT3 pathway has been pinpointed in mouse studies as a key regulator of myeloid suppression, in human DC this is less well established. We studied the effects of STAT3 inhibition on the suppression of monocyte-derived DC differentiation mediated by a short-list of four predominant suppressive factors and found that pharmacological STAT3 inhibition could only counteract the effects of IL-6. Accordingly, in testing a panel of supernatants derived from 11 cell lines representing various types of solid tumors, STAT3 inhibition only modestly affected the suppressive effects of a minority of supernatants. Importantly, combined interference in the STAT3 and p38 pathways completely prevented inhibition of DC differentiation by all tested supernatants and effected superior DC function, evidenced by increased allogeneic T cell reactivity with elevated IL-12p70/IL-10 ratios and Th1 skewing. Combined STAT3 and p38 inhibition also afforded superior protection against the suppressive effects of primary glioma and melanoma supernatants and induced a shift from CD14+cells to CD1a+cells in metastatic melanoma single-cell suspensions, indicating a potential for improved DC differentiation in the tumor microenvironment. We conclude that combined interference in the STAT3 and p38 MAPK signaling pathways is a promising approach to overcome tumor-induced inhibitory signaling in DC precursors and will likely support clinical immunotherapeutic strategies.

ABSTRACTWhereas TLR9 agonists are recognized as powerful stimulators of antitumor immunity, GM-CSF has had mixed reviews. In previously reported randomized trials we assessed the effects of local immune modulation in early-stage melanoma with CpG-B alone or with GM-CSF. Here we discuss the added value of GM-CSF and show sex-related differences.

ABSTRACTPurpose: Local tumor ablation through irreversible electroporation (IRE) may offer a novel therapeutic option for locally advanced pancreatic cancer (LAPC). It may also serve as a means of in vivovaccination. To obtain evidence of the induction of systemic antitumor immunity following local IRE-mediated ablation, we performed an explorative immune monitoring study. Methods: In ten patients enrolled in a clinical trial exploring the safety, feasibility, and efficacy of percutaneous image-guided IRE in LAPC, we determined the frequency and activation state of lymphocytic and myeloid subsets in pre- and post-treatment peripheral blood samples using flow cytometry. Tumor-specific systemic T cell responses to the pancreatic cancer associated antigen Wilms Tumor (WT)1 were determined after in vitrostimulation in an interferon-y enzyme-linked immunospot assay (Elispot), at baseline and at 2 weeks and 3 months after IRE. Results: Our data showed a transient decrease in systemic regulatory T cells (Treg) and a simultaneous transient increase in activated PD-1+T cells, consistent with the temporary reduction of tumor-related immune suppression after the IRE procedure. Accordingly, we found post-IRE boosting of a pre-existing WT1 specific T cell response in two out of three patients as well as the de novoinduction of these responses in another two patients. There was a trend for these WT1 T cell responses to be related to longer overall survival (p= .055). Conclusions: These findings are consistent with a systemic and tumor-specific immune stimulatory effect of IRE and support the combination of percutaneous IRE with therapeutic immune modulation.

ABSTRACTIn patients with cancer, the functionality of Dendritic Cells (DC) is hampered by high levels of tumor-derived suppressive cytokines, which interfere with DC development and maturation. Poor DC development can limit the efficacy of immune checkpoint blockade and in vivovaccination approaches. Interference in intracellular signaling cascades downstream from the receptors of major tumor-associated suppressive cytokines like IL-10 and IL-6, might improve DC development and activation, and thus enhance immunotherapy efficacy. We performed exploratory functional screens on arrays consisting of >1000 human kinase peptide substrates to identify pathways involved in DC development and its inhibition by IL-10 or IL-6. The resulting alterations in phosphorylation of the kinome substrate profile pointed to glycogen-synthase kinase-3β (GSK3β) as a pivotal kinase in both DC development and suppression. GSK3β inhibition blocked human DC differentiation in vitro, which was accompanied by decreased levels of IL-12p70 secretion, and a reduced capacity for T cell priming. More importantly, adenoviral transduction of monocytes with a constitutively active form of GSK3β induced resistance to the suppressive effects of IL-10 and melanoma-derived supernatants alike, resulting in improved DC development, accompanied by up-regulation of co-stimulatory markers, an increase in CD83 expression levels in mature DC, and diminished release of IL-10. Moreover, adenovirus-mediated intratumoral manipulation of this pathway in an in vivomelanoma model resulted in DC activation and recruitment, and in improved immune surveillance and tumor control. We propose the induction of constitutive GSK3β activity as a novel therapeutic means to bolster DC functionality in the tumor microenvironment.

ABSTRACTThough Vγ9Vδ2-T cells constitute only a small fraction of the total T cell population in human peripheral blood, they play a vital role in tumor defense and are therefore of major interest to explore for cancer immunotherapy. Vγ9Vδ2-T cell-based cancer immunotherapeutic approaches developed so far have been generally well tolerated and were able to induce significant clinical responses. However, overall results were inconsistent, possibly due to the fact that these strategies induced systemic activation of Vγ9Vδ2-T cells without preferential accumulation and targeted activation in the tumor. Here we show that a novel bispecific nanobody-based construct targeting both Vγ9Vδ2-T cells and EGFR induced potent Vγ9Vδ2-T cell activation and subsequent tumor cell lysis both in vitroand in an in vivomouse xenograft model. Tumor cell lysis was independent of KRASand BRAFtumor mutation status and common Vγ9Vδ2-T cell receptor sequence variations. In combination with the conserved monomorphic nature of the Vγ9Vδ2-TCR and the facile replacement of the tumor-specific nanobody, this immunotherapeutic approach can be applied to a large group of cancer patients.

ABSTRACTPrevious vaccination studies in patients with castration-resistant prostate cancer (CRPC) showed improved survival without prolongation of progression-free survival (PFS). This might be explained by enhanced efficacy of subsequent therapies because of heightened immune status. We therefore evaluated the efficacy of chemotherapy in CRPC patients after immunotherapy. We retrospectively analyzed 28 patients who were treated with ipilimumab and GVAX, an allogeneic vaccine, and 21 patients who were randomized to GVAX or no vaccination. To study whether immune status was related to the efficacy of chemotherapy, frequencies of myeloid and lymphocyte subsets were determined. Of 28 patients treated with GVAX and ipilimumab, 23 patients received docetaxel and 13 patients mitoxantrone. Median PFS after docetaxel was 6.4 mo (range 0.8–11.2), while median PFS after mitoxantrone was markedly longer than expected (4.8 mo; range 1.4–13.7). High CD8+ICOS+Tcell/Treg and pDC/mMDSC ratios were associated with relatively long PFS after mitoxantrone, suggesting a correlation between activated immune status and benefit of mitoxantrone. Analysis of 21 patients, randomized to GVAX or not, revealed a median PFS after docetaxel of 9.9 mo for vaccinated patients and 7.1 mo for unvaccinated patients. Interestingly, PFS after mitoxantrone (n = 14) was significantly longer in vaccinated patients as compared to controls (5.9 vs. 1.6 mo, p = 0.0048). In conclusion, mitoxantrone seems more effective in CRPC patients after immunotherapy, which may be related to the immune-stimulating effect of mitoxantrone in patients with heightened antitumor immunity. As this was a retrospective study with limited sample size, prospective studies are warranted to definitively show proof of principle.

A number of studies point to an aberrant differentiation and accumulation of CD14+PD-L1+M2-macrophage-like cells in the microenvironment of cervical cancer, which promote immunosuppressive conditions and are associated with tumor invasion, angiogenesis and metastasis. Therapeutic targeting of these macrophages may tip the balance in favor of antitumor immunity. Cervical cancer is the fourth most common cancer among women worldwide and is caused by a persistent infection and subsequent integration of high-risk types of the human papillomavirus. Continuous expression of the viral oncoproteins E6 and E7 has been shown essential to maintain the transformed state of infected keratinocytes. As these non-self oncoproteins are immunogenic, cervical cancer requires a highly immune suppressed tumor microenvironment to metastasize through lymphovascular space invasion (LVSI) to the pelvic tumor-draining lymph nodes (TDLN). Unraveling the mechanisms underlying this immune suppression may uncover novel therapeutic targets aimed at loco-regional control of cervical cancer.

CD1d-restricted invariant natural killer T cells (iNKT) constitute an important immunoregulatory T-cell subset involved in the induction of antitumor immune responses. Here, we provide a view on the recent observation that Vγ9Vδ2-T cells, through trogocytosis of CD1d-containing membrane fragments, have the capacity to act as antigen presenting cells for iNKT.

The generation and loading of dendritic cells (DC) ex-vivofor tumor vaccination purposes is laborious and costly. Direct intradermal (i.d.) administration of tumor-associated antigens could be an attractive alternative approach, provided that efficient uptake and cross-presentation by appropriately activated skin DCs can be achieved. Here, we compare the efficiency of i.d. delivery of relatively small apoptotic blebs (diameter ∼0.1–1 μm) derived from MART-1 transduced acute myeloid leukemia (AML) HL60 cells, to that of larger apoptotic cell remnants (ACR; 2–10 μm) in a physiologically highly relevant human skin explant model. Injection of either fluorescently-labelled ACRs or blebs alone did not affect the number or distribution of migrated DC subsets from skin biopsies after 48 hours, but resulted in a general up-regulation of the co-stimulatory molecules CD83 and CD86 on skin DCs that had ingested apoptotic material. We have previously shown that i.d. administration of GM-CSF and IL-4 resulted in preferential migration of a mature and highly T cell-stimulatory CD11hiCD1a+CD14−dermal DC subset. Here, we found that co-injection of GM-CSF and IL-4 together with either ACRs or blebs resulted in uptake efficiencies within this dermal DC subset of 7.6% (±6.1%) and 19.1% (±15.9%), respectively, thus revealing a significantly higher uptake frequency of blebs (P< 0.02). Intradermal delivery of tumor-derived blebs did not affect the T-cell priming and TH-skewing abilities of migratory skin DC. Nevertheless, in contrast to i.d. administration of ACR, the injection of blebs lead to effective cross-presentation of MART-1 to specific CD8+effector T cells. We conclude that apoptotic bleb-based vaccines delivered through the skin may offer an attractive, and broadly applicable, cancer immunotherapy.

Interleukin-21 (IL-21) has been described as a potent stimulator of antitumor T-cell immunity, but also of autoimmune reactions and oncogenesis. Antigen presenting cells genetically modified to release IL-21 allow for the expansion of tumor-specific T cells exhibiting favorable effector and growth characteristics and a minimal risk of detrimental side effects.

Active immunotherapy may prevent the relapse of acute myeloid leukemia (AML) by inducing leukemia-specific T cells. Here, we investigated whether Wilms’ tumor 1 (WT1) and preferentially expressed antigen in melanoma (PRAME)-specific T cells could be induced upon the priming of healthy donor- and AML patient-derived T cells with HLA-A2-matched, peptide-loaded allogeneic dendritic cells. AML-reactive, tetramer (Tm)-binding and interferon-producing, cytotoxic T lymphocytes specific for PRAME could readily be isolated from healthy individuals and maintained in culture. In this setting, priming efficacy was significantly higher for PRAME than for WT1. The priming of T cells from patient-derived material proved to be near-to-impossible: No leukemia-associated antigen (LAA)-specific T cell could be primed in 4 patients that had recently achieved a complete response (CR), and in only 1 out of 3 patients exhibiting a sustained CR we did observe WT1-specific T cells, though with a low frequency. These findings suggest that the functionality and/or repertoire of T cells differ in healthy subjects and AML patients in CR, and may have repercussions for the implementation of active vaccination approaches against AML.

Interleukin (IL)-10 is a major cancer-related immunosuppressive factor, exhibiting a unique ability to hamper the maturation of dendritic cells (DCs). We have previously reported that IL-10 induces the conversion of activated, migratory CD1a+DCs found in the human skin to CD14+CD141+macrophage-like cells. Here, as a model of tumor-conditioned DC maturation, we functionally assessed CD14-and CD14+DCs that matured in vitro upon exposure to IL-10. IL-10-induced CD14+DCs were phenotypically characterized by a low maturation state as well as by high levels of BDCA3 and DC-SIGN, and as such they closely resembled CD14+cells infiltrating melanoma metastases. Compared with DC matured under standard conditions, CD14+DCs were found to express high levels of B7-H1 on the cell surface, to secrete low levels of IL-12p70, to preferentially induce TH2 cells, to have a lower allogeneic TH cell and tumor antigen-specific CD8+T-cell priming capacity and to induce proliferative T-cell anergy. In contrast to their CD14+counterparts, CD14-monocyte-derived DCs retained allogeneic TH priming capacity but induced a functionally anergic state as they completely abolished the release of effector cytokines. Transcriptional and cytokine release profiling studies indicated a more profound angiogenic and pro-invasive signature of CD14+DCs as compared with DCs matured in standard conditions or CD14−DCs matured in the presence of IL-10. Importantly, signal transducer and activator of transcription 3 (STAT3) depletion by RNA interference prevented the development of the IL-10-associated CD14+phenotype, allowing for normal DC maturation and providing a potential means of therapeutic intervention.