Audencel is a dendritic cell (DC)-based cellular cancer immunotherapy against glioblastoma multiforme (GBM). It is characterized by loading of DCs with autologous whole tumor lysate and in vitro maturation via “danger signals”. The recent phase II “GBM-Vax” trial showed no clinical efficacy for Audencel as assessed with progression-free and overall survival in all patients. Here we present immunological research accompanying the trial with a focus on immune system factors related to outcome and Audencel’s effect on the immune system. Methodologically, peripheral blood samples (from apheresis before Audencel or venipuncture during Audencel) were subjected to functional characterization via enzyme-linked immunospot (ELISPOT) assays connected with cytokine bead assays (CBAs) as well as phenotypical characterization via flow cytometry and mRNA quantification. GBM tissue samples (from surgery) were subjected to T cell receptor sequencing and immunohistochemistry. As results we found: Patients with favorable pre-existing anti-tumor characteristics lived longer under Audencel than Audencel patients without them. Pre-vaccination blood CD8+ T cell count and ELISPOT Granzyme B production capacity in vitro upon tumor antigen exposure were significantly correlated with overall survival. Despite Audencel’s general failure to induce a significant clinical response, it nevertheless seemed to have an effect on the immune system. For instance, Audencel led to a significant up-regulation of the Th1-related immunovariables ELISPOT IFNγ, the transcription factor T-bet in the blood and ELISPOT IL-2 in a dose-dependent manner upon vaccination. Post-vaccination levels of ELISPOT IFNγ and CD8+ cells in the blood were indicative of a significantly better survival. In summary, Audencel failed to reach an improvement of survival in the recent phase II clinical trial. No clinical efficacy was registered. Our concomitant immunological work presented here indicates that outcome under Audencel was influenced by the state of the immune system. On the other hand, Audencel also seemed to have stimulated the immune system. Overall, these immunological considerations suggest that DC immunotherapy against glioblastoma should be studied further – with the goal of translating an apparent immunological response into a clinical response. Future research should concentrate on investigating augmentation of immune reactions through combination therapies or on developing meaningful biomarkers.Electronic supplementary materialThe online version of this article (10.1186/s40478-018-0621-2) contains supplementary material, which is available to authorized users.
T cells in human skin play an important role in the immune defense against pathogens and tumors. T cells are present already in fetal skin, where little is known about their cellular phenotype and biological function. Using single-cell analyses, we identified a naive T cell population expressing αβ and γδ T cell receptors (TCRs) that was enriched in fetal skin and intestine but not detected in other fetal organs and peripheral blood. TCR sequencing data revealed that double-positive (DP) αβγδ T cells displayed little overlap of CDR3 sequences with single-positive αβ T cells. Gene signatures, cytokine profiles and in silico receptor–ligand interaction studies indicate their contribution to early skin development. DP αβγδ T cells were phosphoantigen responsive, suggesting their participation in the protection of the fetus against pathogens in intrauterine infections. Together, our analyses unveil a unique cutaneous T cell type within the native skin microenvironment and point to fundamental differences in the immune surveillance between fetal and adult human skin.
Glioblastoma is the most dangerous brain cancer. One reason for glioblastoma's aggressiveness are glioblastoma stem‐like cells. To target them, a number of markers have been proposed (CD133, CD44, CD15, A2B5, CD36, CXCR4, IL6R, L1CAM, and ITGA6). A comprehensive study of co‐expression patterns of them has, however, not been performed so far. Here, we mapped the multidimensional co‐expression profile of these stemness‐associated molecules. Gliomaspheres – an established model of glioblastoma stem‐like cells – were used. Seven different gliomasphere systems were subjected to multicolor flow cytometry measuring the nine markers CD133, CD44, CD15, A2B5, CD36, CXCR4, IL6R, L1CAM, and ITGA6 all simultaneously based on a novel 9‐marker multicolor panel developed for this study. The viSNE dimensionality reduction algorithm was applied for analysis. All gliomaspheres were found to express at least five different glioblastoma stem‐like cell markers. Multi‐dimensional analysis showed that all studied gliomaspheres consistently harbored a cell population positive for the molecular signature CD44+/CD133+/ITGA6+/CD36+. Glioblastoma patients with an enrichment of this combination had a significantly worse survival outcome when analyzing the two largest available The Cancer Genome Atlas datasets (MIT/Harvard Affymetrix: P = 0.0015, University of North Carolina Agilent: P = 0.0322). In sum, we detected a previously unknown marker combination – demonstrating feasibility, usefulness, and importance of high‐dimensional gliomasphere marker combinatorics.
Glioblastoma is the most prevalent and aggressive brain cancer. With a median overall survival of ~15–20 months under standard therapy, novel treatment approaches are desperately needed. A recent phase II clinical trial with a personalized immunotherapy based on tumor lysate-charged dendritic cell (DC) vaccination, however, failed to prolong survival. Here, we investigated tumor tissue from trial patients to explore glioblastoma survival-related factors. We followed an innovative approach of combining mass spectrometry-based quantitative proteomics (n = 36) with microRNA sequencing plus RT-qPCR (n = 38). Protein quantification identified, e.g., huntingtin interacting protein 1 (HIP1), retinol-binding protein 1 (RBP1), ferritin heavy chain (FTH1) and focal adhesion kinase 2 (FAK2) as factor candidates correlated with a dismal prognosis. MicroRNA analysis identified miR-216b, miR-216a, miR-708 and let-7i as molecules potentially associated with favorable tissue characteristics as they were enriched in patients with a comparably longer survival. To illustrate the utility of integrated miRNomics and proteomics findings, focal adhesion was studied further as one example for a pathway of potential general interest.Taken together, we here mapped possible drivers of glioblastoma outcome under immunotherapy in one of the largest DC vaccination tissue analysis cohorts so far—demonstrating usefulness and feasibility of combined proteomics/miRNomics approaches. Future research should investigate agents that sensitize glioblastoma to (immuno)therapy—potentially building on insights generated here.
The adult human skin contains a vast number of T cells that are essential for skin homeostasis and pathogen defense. T cells are first observed in the skin at the early stages of gestation, however, our understanding of their contribution to early immunity has been limited by their low abundance and lack of comprehensive methodologies for their assessment. Here, we describe a new workflow for isolating and expanding significant amounts of T cells from fetal human skin. Using multiparametric flow cytometry and in situ immunofluorescence we found a large population with a naive and small populations with a memory and regulatory phenotype. Their molecular state was characterized using single-cell transcriptomics and TCR repertoire profiling. Importantly, culture of total fetal skin biopsies facilitated T cell expansion without a substantial impact on their phenotype, a major prerequisite for subsequent functional assays. Collectively, our experimental approaches and data advance the understanding of fetal skin immunity and potential use in future therapeutic interventions.
Skin resident T cells provide immediate immunologic responses at their specific location and play a role in the pathogenesis of skin diseases such as psoriasis. Recently, IL-9–producing T cells were described as a major T-cell subtype present in the skin, but knowledge on the biology and in situ regulation of this T-cell subtype is scarce. Here, we investigated the cytokine influence on skin T cells with focus on IL-9–producing T cells because a better understanding of their biology may identify novel therapeutic approaches. Healthy human skin biopsies were cultured either in the presence of IL-2, IL-4, and TGF-β [T helper (T h )9–promoting condition (T h 9-PC)] or IL-2 and IL-15 [standard condition (SC)]. Paired analysis of enzymatically isolated skin T cells and emigrated T cells after 4 wk of skin culture showed significant alterations of T-cell phenotypes, cytokine production, and IL-9–producing T-cell frequency. RNA sequencing analysis revealed differentially regulated pathways and identified CXCL8 and CXCL13 as top up-regulated genes in T h 9-PC compared with SC. Functionally supernatant of stimulated skin-derived T cells, CXCL8 and CXCL13 increased neutrophil survival. We report that the cytokine environment alters skin-derived T-cell phenotype and functional properties.—Kienzl, P., Polacek, R., Reithofer, M., Reitermaier, R., Hagenbach, P., Tajpara, P., Vierhapper, M., Gschwandtner, M., Mildner, M. Jahn-Schmid, B., Elbe-Bürger, A. The cytokine environment influence on human skin–derived T cells.
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