Rebekka Wehner

Ovarian cancer (OC) is a heterogeneous tumor entity with accumulated ascitic fluid in the peritoneal cavity, especially in advanced tumors. In general, a high immune cell infiltration has a favorable effect on OC patients’ outcomes. However, the composition of immune cells within the individual compartments of OC-associated locations may differ in their impact on patient prognosis. Therefore, we comprehensively investigated immune cell frequencies in matched peripheral blood, ascites, and tumor samples of 24 high-grade serous OC patients by flow cytometry and associated them with clinical parameters. Immune cell analysis demonstrated that the general immune cell infiltration was comparable between the three investigated compartments, with decreased proportions of CD8+ T cells in advanced stage OC. In addition, immune cell subsets varied significantly in their differentiation and phenotypic marker expression. In peripheral blood, classical monocytes, mature natural killer (NK) cells with cytotoxic potential (CD57+, CD16+, NKG2D+), and less differentiated T cells were more frequent. On the contrary, dendritic cells, and NKp46+ NK cells were prevalent in ascites. In OC tissues, high frequencies of immature neutrophils, CD16- NK cells, and effector memory T cells were found, although the intratumoral T cell frequency was significantly reduced compared to the two liquid samples. Additionally, T cell profiling showed high expression of one or multiple activating and inhibitory receptors in tumor samples. In particular, significant positive correlations of CD127+ CD8+ T cells among all three compartments were shown. Our results provide evidence that a higher proportion of peripheral CD127+ CD8+ T cells, which are memory T cells with low granzyme B production, was a prognostic biomarker for unfavorable progression-free survival of high-grade OC patients, independent of FIGO stage III/IV or residual tumor after surgery.

Background/Objectives: Peripheral neuroblastic tumors (pNT) are a biologically heterogeneous group of embryonal tumors that derive from the neural crest and affect the sympathetic nervous system. So far, little is known about the complex immune landscape in these rare childhood cancers. Methods: We focused on the immune cell infiltrate of treatment-naïve pNT from 24 patients, including high-risk neuroblastoma (HR-NBL), non-high-risk neuroblastoma (NHR-NBL), ganglioneuroblastoma (GNBL), and rare ganglioneuroma (GN). To gain novel insights into the immune architecture of these pNT subtypes, we used multiplex immunohistochemistry, multispectral imaging, and algorithm-based data evaluation to detect and characterize T cells, B cells, neutrophils, macrophages, and tertiary lymphoid structures (TLS). Results: The majority of the investigated tumor-infiltrating immune cells were macrophages and T cells. Their detailed phenotypic characterization revealed high proportions of M2-like macrophages as well as activated GrzB⁺ CD8⁺ and PD-1⁺ T lymphocytes. Proportions of these T cell phenotypes were significantly increased in GN compared to HR-NBL, NHR-NBL, or GNBL. In addition, TLS occurred in 11 of 24 patients, independent of immune cell frequencies in the whole tissues. Interestingly, all GN, most GNBL, but only a few NBL contained TLS. We distinguished between three TLS maturation stages that were present irrespective of the pNT subtype. The majority belonged to mature TLS of the primary follicle state. Mature LAMP3⁺ dendritic cells were also found, predominantly in T cell zones of TLS. Furthermore, TLS presence identified pNT patients with significantly prolonged progression-free survival in contrast to all other analyzed immunological features. Conclusions: We propose TLS to be a potential prognostic marker for pNT to predict patient outcomes.

Bone marrow mesenchymal stromal cells (MSCs) have been described as potent regulators of T-cell function, though whether they could impede the effectiveness of immunotherapy against acute myeloid leukemia (AML) is still under investigation. We examine whether they could interfere with the activity of leukemia-specific clonal cytotoxic T-lymphocytes (CTLs) and chimeric antigen receptor (CAR) T cells, as well as whether the immunomodulatory properties of MSCs could be associated with the induction of T-cell senescence. Co-cultures of leukemia-associated Wilm’s tumor protein 1 (WT1) and tyrosine-protein kinase transmembrane receptor 1 (ROR1)-reactive CTLs and of CD123-redirected switchable CAR T cells were prepared in the presence of MSCs and assessed for cytotoxic potential, cytokine secretion, and expansion. T-cell senescence within functional memory sub-compartments was investigated for the senescence-associated phenotype CD28⁻CD57⁺ using unmodified peripheral blood mononuclear cells. We describe inhibition of expansion of AML-redirected switchable CAR T cells by MSCs via indoleamine 2,3-dioxygenase 1 (IDO-1) activity, as well as reduction of interferon gamma (IFNγ) and interleukin-2 (IL-2) release. In addition, MSCs interfered with the secretory potential of leukemia-associated WT1- and ROR1-targeting CTL clones, inhibiting the release of IFNγ, tumor necrosis factor alpha, and IL-2. Abrogated T cells were shown to retain their cytolytic activity. Moreover, we demonstrate induction of a CD28^loCD27^loCD57⁺KLRG1⁺ senescent T-cell phenotype by MSCs. In summary, we show that MSCs are potent modulators of anti-leukemic T cells, and targeting their modes of action would likely be beneficial in a combinatorial approach with AML-directed immunotherapy.

Dendritic cells (DCs) play a key role in the orchestration of antitumor immunity. Activated DCs efficiently enhance antitumor effects mediated by natural killer cells and T lymphocytes. Conversely, tolerogenic DCs essentially contribute to an immunosuppressive tumor microenvironment. Thus, DCs can profoundly influence tumor progression and clinical outcome of tumor patients. To gain novel insights into the role of human DCs in pancreatic ductal adenocarcinoma (PDAC), we explored the frequency, spatial organization, and clinical significance of conventional DCs type 1 (cDC1s) and type 2 (cDC2s) and plasmacytoid DCs (pDCs) in primary PDAC tissues. A higher density of whole tumor area (WTA)- and tumor stroma (TS)-infiltrating cDC1s was significantly associated with better disease-free survival (DFS). In addition, an increased frequency of intraepithelial tumor-infiltrating cDC2s was linked to better DFS and overall survival (OS). Furthermore, an increased density of WTA- and TS-infiltrating pDCs tended to improve DFS. Moreover, a higher frequency of WTA- and TS-infiltrating cDC1s and pDCs emerged as an independent prognostic factor for better DFS and OS. These findings indicate that tumor-infiltrating DCs can significantly influence the clinical outcome of PDAC patients and may contribute to the design of novel treatment options that target PDAC-infiltrating DCs.

The administration of antibodies blocking the immune checkpoint molecules programmed cell death protein 1 (PD-1) or programmed cell death 1 ligand 1 (PD-L1) has evolved as a very promising treatment option for cancer patients. PD-1/PD-L1 inhibition has significantly enhanced expansion, cytokine secretion, and cytotoxic activity of CD4⁺ and CD8⁺ T lymphocytes, resulting in enhanced antitumor responses. Anti-PD-1 or anti-PD-L1 therapy has induced tumor regression and improved clinical outcome in patients with different tumor entities, including melanoma, non-small-cell lung cancer, and renal cell carcinoma. These findings led to the approval of various anti-PD-1 or anti-PD-L1 antibodies for the treatment of tumor patients. However, the majority of patients have failed to respond to this treatment modality. Comprehensive immune monitoring of clinical trials led to the identification of potential biomarkers distinguishing between responders and non-responders, the discovery of modes of treatment resistance, and the design of improved immunotherapeutic strategies. In this review article, we summarize the evolving landscape of biomarkers for anti-PD-1 or anti-PD-L1 therapy.

Targeting the immune checkpoint receptors cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), or programmed cell death 1 ligand 1 (PD-L1) represents a very attractive treatment modality for tumor patients. The administration of antibodies against these receptors can promote efficient antitumor effects and can induce objective clinical responses in about 20–40% patients with various tumor types, accompanied by improved survival. Based on their therapeutic efficiency, several antibodies have been approved for the treatment of tumor patients. However, many patients do not respond to checkpoint inhibitor therapy. Therefore, the identification of biomarkers is required to guide patient selection for this treatment modality. Here, we summarize recent studies investigating the PD-L1 expression or mutational load of tumor tissues as well as the frequency and phenotype of immune cells in tumor patients prior to and during CTLA-4 or PD-1/PD-L1 inhibitor treatment.

Chemotherapy is an important treatment modality for many patients with advanced cancer. Recent data revealed that certain chemotherapeutic agents differentially affect maturation, cytokine production and T‐cell stimulatory capacity of dendritic cells (DCs), which play a crucial role in the induction of antitumor immunity. Whereas most reports are based on mouse or human monocyte‐derived DCs, studies investigating the direct effect of chemotherapeutic drugs on native human DCs are rather limited. Here, we evaluated the impact of various chemotherapeutic drugs on the immunostimulatory properties of 6‐sulfo LacNAc⁺ (slan) DCs, representing a major subpopulation of human blood DCs. Because of their various antitumor effects, slanDCs may essentially contribute to the immune defense against tumors. We demonstrated that doxorubicin and vinblastine significantly impair the release of tumor necrosis factor‐α, interleukin (IL)‐6 and IL‐12 by slanDCs. Functional data revealed that both drugs inhibit slanDC‐mediated proliferation of T lymphocytes and their capacity to differentiate naive CD4⁺ T cells into proinflammatory T‐helper type I cells. Furthermore, these agents markedly suppressed the ability of slanDCs to stimulate interferon‐γ secretion by natural killer (NK) cells. In contrast, paclitaxel, mitomycin C and methotrexate sustained the ability of slanDCs to produce proinflammatory cytokines and their potential to activate T‐lymphocytes and NK cells. These results indicate that doxorubicin and vinblastine impair the ability of native human DCs to stimulate important immune effector cells, whereas methotrexate, mitomycin C and paclitaxel maintain their immunostimulatory properties. These novel findings may have implications for the design of treatment modalities for tumor patients combining immunotherapeutic strategies and chemotherapy.

Mesenchymal stromal cells (MSCs) have emerged as promising candidates for regenerative therapies, including tissue engineering. Recently it has been reported that engineered extracellular matrix (ECM) components support the differentiation of MSCs into osteocytes and chondrocytes, indicating that ECM components may represent attractive carriers for MSC transplants to repair damaged tissues. However, little is known about the impact of engineered ECM components on the immunosuppressive properties of MSCs, which may essentially contribute to the prevention of allogeneic MSC transplant rejection. In the present study, we explored the potential of fibronectin, fibrillar collagen I, tropocollagen and collagen I/heparin to influence the immunosuppressive capacities of MSCs. We found that these ECM components do not modulate the capability of MSCs to inhibit the proliferation of anti‐CD3/anti‐CD28 antibody‐stimulated CD4⁺ and CD8⁺ T cells and of lymphocytes in a mixed lymphocyte reaction. In addition, the potential of MSCs to impair the production of immunostimulatory IL‐12 and to improve the release of immunosuppressive IL‐10 by 6‐sulpho LacNAc⁺ (slan) dendritic cells (DCs), representing a pro‐inflammatory subset of human blood DCs, was not altered by the ECM components. Furthermore, ECM components do not influence the ability of MSCs to inhibit the slanDC‐induced proliferation of CD4⁺ T cells. In conclusion, the used engineered ECMs maintain important immunosuppressive properties of MSCs, which support their suitablility as carriers for MSC transplants in tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.

Prostate cancer (PCa) is the most common noncutaneous cancer diagnosis and the second leading cause of cancer-related deaths among men in the United States. Effective treatment modalities for advanced metastatic PCa are limited. Immunotherapeutic strategies based on T cells and antibodies represent interesting approaches to prevent progression from localized to advanced PCa and to improve survival outcomes for patients with advanced disease. CD8⁺ cytotoxic T lymphocytes (CTLs) efficiently recognize and destroy tumor cells. CD4⁺ T cells augment the antigen-presenting capacity of dendritic cells and promote the expansion of tumor-reactive CTLs. Antibodies mediate their antitumor effects via antibody-dependent cellular cytotoxicity, activation of the complement system, improving the uptake of coated tumor cells by phagocytes, and the functional interference of biological pathways essential for tumor growth. Consequently, several tumor-associated antigens (TAAs) have been identified that represent promising targets for T cell- or antibody-based immunotherapy. These TAAs comprise proteins preferentially expressed in normal and malignant prostate tissues and molecules which are not predominantly restricted to the prostate, but are overexpressed in various tumor entities including PCa. Clinical trials provide evidence that specific immunotherapeutic strategies using such TAAs represent safe and feasible concepts for the induction of immunological and clinical responses in PCa patients. However, further improvement of the current approaches is required which may be achieved by combining T cell- and/or antibody-based strategies with radio-, hormone-, chemo- or antiangiogenic therapy.

Purpose: To evaluate whether immunotherapy based on adoptively transferred cytotoxic T-cells (CTL) can improve the antitumour efficacy of irradiation. Material and methods: The experiments were performed using the human squamous cell carcinoma line UT-SCC-15, which expresses human leukocyte antigen (HLA)-A2. The UT-SCC-15 cell-mediated activation of JB4 CTL in terms of interferon (IFN)-γ secretion and cytotoxic potential was determined by enzyme-linked immunosorbent assay and chromium release assay, the perforin content of JB4 cells by flow cytometry. In vivo, tumours were irradiated with 14 Gy. Subsequently, JB4 CTL were injected intra- and peritumourally. Volume doubling times were calculated as a marker of tumour growth delay. Results: UT-SCC-15 tumour cells were well recognized by JB4 CTL in vitro, as indicated by profound IFN-γ secretion and tumour cell lysis. This response was completely abrogated in the presence of an anti-HLA-A2 antibody. In vivo, adoptive transfer of JB4 CTL after irradiation did not delay tumour growth in comparison to irradiation alone. As a possible underlying mechanism, a loss of perforin content and cytolytic function of the CTL in the absence of interleukin (IL)- 2 or IL-15 was found in vitro. Conclusion: HLA-A2-alloreactive JB4 cells efficiently recognize and destroy UT-SCC-15 tumour cells in vitro. However, the intratumoural application of JB4 cells after irradiation does not enhance the in vivo effect of radiotherapy alone, which might be caused by the reduced cytotoxic potential of JB4 cells in the absence of IL-2 or IL-15. Thus, co-administration of these cytokines might improve the efficacy of combined irradiation and CTL treatment.