Allogeneic NK cells induce the in vitro activation of monocyte-derived and conventional type-2 dendritic cells and trigger an inflammatory response under cancer-associated conditions.

Natural killer (NK) cells are innate lymphocytes capable to recognize and kill virus-infected and cancer cells. In the past years, the use of allogeneic NK cells as anti-cancer therapy gained interest due to their ability to induce graft-versus-cancer responses without causing graft-versus-host disease and multiple protocols have been developed to produce high numbers of activated NK cells. While the ability of these cells to mediate tumor kill has been extensively studied, less is known about their capacity to influence the activity of other immune cells that may contribute to a concerted anti-tumor response in the tumor microenvironment (TME). In this study, we analyzed how an allogeneic off-the-shelf cord blood stem cell-derived NK-cell product influenced the activation of dendritic cells (DC). Crosstalk between NK cells and healthy donor monocyte-derived DC (MoDC) resulted in the release of IFNγ and TNF, MoDC activation, and the release of the T-cell-recruiting chemokines CXCL9 and CXCL10. Moreover, in the presence of prostaglandin-E2, NK cell/MoDC crosstalk antagonized the detrimental effect of IL-10 on MoDC maturation leading to higher expression of multiple (co-)stimulatory markers. The NK cells also induced activation of conventional DC2 (cDC2) and CD8+ T cells, and the release of TNF, GM-CSF, and CXCL9/10 in peripheral blood mononuclear cells of patients with metastatic colorectal cancer. The activated phenotype of MoDC/cDC2 and the increased release of pro-inflammatory cytokines and T-cell-recruiting chemokines resulting from NK cell/DC crosstalk should contribute to a more inflamed TME and may thus enhance the efficacy of T-cell-based therapies.

Oncolytic adenovirus ORCA-010 increases the type 1 T cell stimulatory capacity of melanoma-conditioned dendritic cells.

Immune checkpoint blockade has resulted in durable responses in patients with metastatic melanoma, but only in a fraction of treated patients. For immune checkpoint inhibitors (ICI) to be effective, sufficient infiltration with tumor-reactive T cells is essential. Oncolytic viruses (OV) selectively replicate in and lyse tumor cells and so induce an immunogenic form of cell death, providing at once a source of tumor-associated (neo)antigens and of danger signals that together induce effective T cell immunity and tumor infiltration. Melanoma-associated suppression of dendritic cell (DC) differentiation effectively hampers OV- or immune checkpoint inhibitor (ICI)-induced anti-tumor immunity, due to a consequent inability to prime and attract anti-tumor effector T cells. Here, we set out to study the effect of ORCA-010, a clinical stage oncolytic adenovirus, on DC differentiation and functionality in the context of human melanoma. In melanoma and monocyte co-cultures, employing a panel of five melanoma cell lines with varying origins and oncogenic mutation status, we observed clear suppression of DC development with apparent skewing of monocyte differentiation to a more M2-macrophage-like state. We established the ability of ORCA-010 to productively infect and lyse the melanoma cells. Moreover, although ORCA-010 was unable to restore DC differentiation, it induced activation and an increased co-stimulatory capacity of monocyte-derived antigen-presenting cells. Their subsequent ability to prime effector T cells with a type I cytokine profile was significantly increased in an allogeneic mixed leukocyte reaction. Our findings suggest that ORCA-010 is a valuable immunotherapeutic agent for melanoma.

'DURVIT': a phase-I trial of single low-dose durvalumab (Medi4736) IntraTumourally injected in cervical cancer: safety, toxicity and effect on the primary tumour- and lymph node microenvironment.

BACKGROUND: Treatment with programmed cell death receptor (PD-1) and programmed cell death ligand 1 (PD-L1) inhibitors is a promising strategy to lift tumour-induced immune response suppression. However, the current systemic treatment often causes autoimmune side effects. In more than 50% of squamous cell cervical cancer, PD-L1 expression is detected. Moreover, we observed high and interrelated rates of PD-L1 positive macrophages and regulatory T cells in metastatic lymph nodes of cervical cancer patients. As cervical cancer in general initially metastasizes to regional lymph nodes, local administration of durvalumab (a PD-L1 checkpoint inhibitor) at an early stage will deliver these antibodies exactly where they are needed, facilitating immune protection. This may result in a clinical benefit while reducing undesirable side effects. METHODS: DURVIT is a non-randomized, single-arm, open-label, phase I study. Three escalating dose levels of intratumourally (i.t.) injected durvalumab will be tested, i.e. 5, 10 and 20 mg (three patients per dose level, with an additional three at the highest tolerated dose). The primary endpoint of this phase-I study is safety. Immune monitoring will consist of flow cytometric, immunohistochemical and functional T cell reactivity testing. The first patient has been included in this trial in November 2017. DISCUSSION: Evidence of safety and biological efficacy of this locally administered checkpoint blockade may expand adjuvant therapy options for cervical cancer patients. Early metastatic spread of cervical cancer cells may thus be controlled in the draining lymph node basin, and beyond, and hopefully delay or even prevent the onset of disease recurrence. TRIAL REGISTRATION: NTR6119 , 1-nov-2016.

An organotypic human melanoma-in-skin model as an in vitro tool for testing Vγ9Vδ2-T cell-based immunotherapy.

Background: Despite considerable advancements in cancer immunotherapy, advanced melanoma still presents a substantial clinical challenge. In an effort to explore treatment options, we examined the immunotherapeutic potential of effector Vγ9Vδ2-T cells in vitro in a three-dimensional (3D) human organotypic melanoma-in-skin (Mel-RhS) model. Materials and methods: Vγ9Vδ2-T cells were introduced into Mel-RhS via intradermal injection and cultured within the tissue microenvironment for up to 3 days. Results: Vγ9Vδ2-T cells remained viable for up to 3 days and were in close proximity to or within tumor nests. Upon Mel-RhS dissociation, a fraction was shown to be decorated by melanoma-associated chondroitin sulfate proteoglycan (MCSP), demonstrating their ability to actively navigate the tumor microenvironment and trogocytose cancer cells. Investigation into the apparent trogocytosis revealed an enhanced activated state of MCSP-decorated Vγ9Vδ2-T cells, evidenced by increased expression levels of 4-1BB, NKp44, programmed cell death protein-1 (PD-1), and programmed death-ligand 1 (PD-L1), compared with their MCSP- counterpart. These findings suggest that Vγ9Vδ2-T cells, upon successfully contacting melanoma cells, actively recognize and acquire MCSP from these malignant cells. Evidence of actual tumor cell elimination, although not significant, was only obtained after preincubation of Mel-RhS with pamidronate, a phosphoantigen-inducing agent, indicating the need for additional T cell receptor-mediated signaling for Vγ9Vδ2-T cells to reach their full oncolytic potential. Conclusions: This study highlights the viability and persistence of Vγ9Vδ2-T cells within the 3D microenvironment, their migratory and antitumor functionality, and the suitability of the model for testing T cell-based therapies, contributing both to the understanding of Vγ9Vδ2-T cell biology and their application in cancer immunotherapy.

The dermis as a portal for dendritic cell-targeted immunotherapy of cutaneous melanoma.

Complete surgical excision at an early stage remains the only curative treatment for cutaneous melanoma with few available adjuvant therapy options. Nevertheless, melanoma is a relatively immunogenic tumor type and particularly amenable to immunotherapeutic approaches. A dense network of cutaneous dendritic cells (DC) may account for the reported efficacy of vaccination through the skin and provide an attractive target for the immunotherapy of melanoma. Several phenotypically distinct DC subsets are discernable in the skin, among others, epidermal Langerhans cells and dermal DC. Upon appropriate activation both subsets can efficiently migrate to melanoma-draining lymph nodes (LN) to prime T cell-mediated responses. Unfortunately, from an early stage, melanoma development is characterized by strong immune suppression, facilitating unchecked tumor growth and spread. Particularly the primary tumor site and the first-line tumor-draining LN, the so-called sentinel LN, bear the brunt of this melanoma-induced immune suppression-and these are exactly the sites where anti-melanoma effector T cell responses should be primed by DC in order to prevent early metastasis. Through local immunopotentiation or through DC-targeted vaccination, the dermis may be utilized as a portal to activate DC and kick-start or boost effective T cell-mediated anti-melanoma immunity, even in the face of this immune suppression.

Effector memory T-cell frequencies in relation to tumour stage, location and HPV status in HNSCC patients.

BACKGROUND: The immune system plays an important role in tumour immune surveillance. Head and neck squamous cell carcinoma patients are often immune compromised. OBJECTIVE: To chart the baseline levels of T-cell subpopulation frequencies in patients with cancer prior to treatment. SUBJECTS AND METHODS: Blood samples of patients were taken at the time of diagnosis, analysed with flowcytometry and compared with blood samples of healthy donors. RESULTS: Compared to healthy donors, a significant shift from naive to effector memory T cells was observed. This effect was most prominent in stage II patients. A similar shift from naive to effector memory T cells was noted in patients with oropharynx or larynx squamous cell carcinomas. Furthermore, the percentage of effector memory and effector T cells was higher in the group of patients with human papillomavirus-positive oropharyngeal squamous cell carcinomas, compared with patients with human papillomavirus-negative tumours, suggestive of virus-induced T-cell activation. CONCLUSION: Here, we provide a simple and easily implementable tool to document T lymphocyte subsets in the peripheral blood of head and neck cancer patients, which might be useful for prognosis and/or therapy response prediction.

Pre-treatment tumor-infiltrating T cells influence response to neoadjuvant chemoradiotherapy in esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) is a disease with dismal treatment outcomes. Response to neoadjuvant chemoradiation (CRT) varies greatly. Although the underlying mechanisms of CRT resistance are not identified, accumulating evidence indicates an important role for local antitumor immunity. To explore the immune microenvironment in relation to response to CRT we performed an in-depth analysis using multiplex immunohistochemistry, flow cytometry and mRNA expression analysis (NanoString) to generate a detailed map of the immunological landscape of pretreatment biopsies as well as peripheral blood mononuclear cells (PBMCs) of EAC patients. Response to CRT was assessed by Mandard's tumor regression grade (TRG), disease-free- and overall survival. Tumors with a complete pathological response (TRG 1) to neoadjuvant CRT had significantly higher tumor-infiltrating T cell levels compared to all other response groups (TRG 2-5). These T cells were also in closer proximity to tumor cells in complete responders compared to other response groups. Notably, immune profiles of near-complete responders (TRG 2) showed more resemblance to non-responders (TRG 3-5) than to complete responders. A high CD8:CD163 ratio in the tumor was associated with an improved disease-free survival. Gene expression analyses revealed that T cells in non-responders were Th2-skewed, while complete responders were enriched in cytotoxic immune cells. Finally, complete responders were enriched in circulating memory T cells. preexisting immune activation enhances the chance for a complete pathological response to neoadjuvant CRT. This information can potentially be used for future patient selection, but also fuels the development of immunomodulatory strategies to enhance CRT efficacy.

4-1BB-mediated expansion affords superior detection of in vivo primed effector memory CD8+ T cells from melanoma sentinel lymph nodes.

We have been studying the re-activation of tumor-associated antigen (TAA)-specific CD8(+) T cells in sentinel lymph nodes (SLN) of melanoma patients upon intradermal administration of the CpG-B oligodeoxynucleotide PF-3512676. To facilitate functional testing of T cells from small SLN samples, high-efficiency polyclonal T cell expansion is required. In this study, SLN cells were expanded via classic methodologies with plate- or bead-bound anti-CD3/CD28 antibodies and with the K562/CD32/4-1BBL artificial APC system (K32/4-1BBL aAPC) and analyzed for responsiveness to common recall or TAA-derived peptides. K32/4-1BBL-expanded T cell populations contained significantly more effector/memory CD8(+) T cells. Moreover, recall and melanoma antigen-specific CD8(+) T cells were more frequently detected in K32/4-1BBL-expanded samples as compared with anti-CD3/CD28-expanded samples. We conclude that K32/4-1BBL aAPC are superior to anti-CD3/CD28 antibodies for the expansion of in vivo-primed specific CD8(+) T cells and that their use facilitates the sensitive monitoring of functional anti-tumor T cell immunity in SLN.

Efficacy of PD-1 blockade in cervical cancer is related to a CD8+FoxP3+CD25+ T-cell subset with operational effector functions despite high immune checkpoint levels.

BACKGROUND: Cervical cancer (CxCa) is mainly a locally invading disease that metastasizes to loco-regional lymph node basins before involving distant organs in more advanced stages. Local immune potentiation of tumor-draining lymph nodes (TDLN) may thus protect against tumor progression. METHODS: To identify therapeutic targets for local immune modulation, multi-parameter flow cytometric T-cell profiling of primary cervical tumors (PT) and TDLN (n = 37) was performed. The in-vitro effect of PD-1 blockade on T-cell reactivity to HPV16 E6 oncoproteins was determined in cultures of TDLN and PT single cell suspensions (n = 19). Also, intracellular cytokine staining (ICS) upon anti-CD3 stimulation was performed in metastatic TDLN (LN+) and PT (n = 7), as well as multiplexed immunofluorescence histochemistry staining (n = 8). RESULTS: Our data revealed elevated rates of activated regulatory T cells (aTregs) and of central or effector memory CD8+ T cells in metastatic TDLN (LN+) as compared to tumor-free TDLN (LN-), and equally high or even higher rates of these subsets in PT. Both memory subsets co-expressed multiple immune checkpoints. PD-1 blockade significantly enhanced detectable E6-specific T-cell responses in 4/5 HPV16+ LN+ and in 1/5 HPV16+ PT. Whereas aTreg rates were higher in anti-PD-1 non-responders, in responders elevated levels of CD8+FoxP3+CD25+ T cells were observed, which correlated with the efficacy of PD-1 blockade (P = 0.018). This subset was characterized by an early effector memory phenotype with particularly high levels of co-expressed PD-1, CTLA-4, TIM-3 and LAG-3 checkpoints, but, rather than exhausted, was shown upon polyclonal activation to produce higher levels of Granzyme-B and effector cytokines as compared to its CD8+FoxP3- counterparts. CONCLUSION: These observations support local PD-(L)1 blockade to interrupt loco-regional immune suppression in CxCa and control metastatic spread to TDLN. Furthermore, our data identify CD8+FoxP3+CD25+ T cells as therapeutic targets, which may also serve as predictive biomarker for PD-(L)1 checkpoint blockade.