Immune assays & imaging

Investigating immunologic mechanisms is a fundamental part of TRON’s translational research. In particular, the efficacy of vaccines and combinatorial therapies to generate, boost and reinvigorate the cellular response against cancers is our focus.  Three specialist teams contribute to this work, both on internal projects and in support of other unit projects.

The Vaccines and Cellular Immunotherapies (VCI) unit designs pre-clinical combination therapeutic studies (using RNA vaccines in liposomes, antibodies, immune checkpoint inhibitors (ICIs), chemo- and radiotherapy, nanoparticles) in syngeneic & xenograft cancer models, performing these with the Preclinical Models team.  Another central component is research performed on primary cultures using 3D air-liquid interface, allowing evaluation of immunomodulatory drugs ex vivo.  The VCI team utilizes a variety of immunological assays such as intracellular cytokine staining, degranulation assay, tetramer staining, and antibody-mediated flow cytometry (up to 18 fluorescent dyes) for the phenotypic and functional characterization of the tumor immune microenvironment including cell-cell interactions and (cytotoxic) T cell functionality.

Our Central Cell Culture acts as a core facility, creating a demand-oriented, broad bank of quality controlled human and animal cells including various tissues and tumor entities, and produce standardized cells pellets for further analyses (NGS, RT-PCR, WB, HLA- typing).  In addition, this team establishes cell-based assays and custom culture methods for various metrics (e.g. ADCC, flow cytometry, immunocytochemistry, generation of sub-clones).

TRON has a variety of imaging platforms allowing the visualization of immune and tumor cells at the cellular and organ level.  Among these (in vivo imaging is performed by the Preclinical Models team), multiplex immunofluorescence allows imaging the spatial distribution of specific proteins and immune cells in tissues. This method is particularly helpful when exploring immune evasion mechanisms and finding potential biomarkers.  In addition, live imaging reveals the temporal dynamics of the immune response.  This is achieved with expertise in advanced histology & cryo-immunofluorescence.


Tools and Platforms

Design immunotherapy (pre-clinical) studies

  • Combination therapy studies (RNA vaccines, anti-tumor antibody, immune checkpoint inhibition, chemo- and local radiotherapy)
  • Characterization of the intratumoral and systemic immnue response

Air-liquid interface culture

  • Explanted primary murine and human tumors

Multicolor flow cytometry assays (max 18 fluorescent dyes)

  • Functional assays (effector cytokine release and degranulation)
  • Cell death (cleaved caspase-3 and AnnexinV/7AAD)
  • Cell proliferation (BrdU, Ki67)
  • Phagocytsis

Multicolor IF (4 colors)

  • Detection of immune cells, proliferating cells, cell death and vasculature in tumors, lymph nodes, spleen, lung and small intestine
  • Tumor microenvironment characterization (hypoxia, vasculature leakiness)

Live imaging

  • Spatiotemporal studies of cell-cell interaction, cell killing and motility

Ex vivo Multiphoton Imaging:

  • Visualization of fluorescently labelled immune cells in explanted lymph nodes and small intestine

Multiplex Immunofluorescence Platform


  • Low order 2-6plex on FFPE samples
  • Akoya Opal TSA-based flurophores for multiplex IHC
  • Automated staining workflow on Leica BOND Rx (30 slides/run) or manual staining


  • Vectra Polaris Multispectral Slide Scannermicroscope
  • Whole slide multispectral imaging up to 7 colors with MOTiF technology
  • Bright-field imaging
  • Fully automated, high-throughput system (80 slides)


  • Phenochart and Inform: Spectrally unmixing images, tissue seg, cell seg phenotyping, scoring, pathology views

Key Contributions and Publications

Salomon N, Vascotto F, Selmi A, Vormehr M, Quinkhardt J, Bukur T, Schrörs B, Löwer M, Diken M, Türeci Ö, Sahin U, Kreiter S. (2020) A liposomal RNA vaccine inducing neoantigen-specific CD4+ T cells augments the antitumor activity of local radiotherapy in mice. Oncoimmunology. 9: 1771925.

Vascotto F, Petschenka J, Walzer KC, Vormehr M, Brkic M, Strobl S, Rösemann R, Diken M, Kreiter S, Türeci Ö, Sahin U. (2019) Intravenous delivery of the toll-like receptor 7 agonist SC1 confers tumor control by inducing a CD8+ T cell response. Oncoimmunology. 8(7):1601480.

Vormehr M, Reinhard K, Blatnik R, Josef K, Beck J, Salomon N, Suchan M, Selmi A, Vascotto F, Zerweck J, Wenschuh H, Diken M. Kreiter S, Türeci Ö,  Riemer AG, Sahin U. (2018) A non-functional neoepitope specific CD8+ T-cell response induced by tumor derived antigen exposure in vivo. Oncoimmunology . 8(3):1553478.

Fiore A, Ugel S, De Sanctis F, Sandri S, Fracasso G, Trovato R, Sartoris S, Solito S, Mandruzzato S, Vascotto F, Hippen KL, Mondanelli G, Grohmann U, Piro G, Carbone C, Melisi D, Lawlor RT, Scarpa A, Lamolinara A, Iezzi M, Fassan M, Bicciato S, Blazar BR, Sahin U, Murray PJ, Bronte V. (2018) Induction of immunosuppressive functions and NF-κB by FLIP in monocytes. Nat. Commun. 9(1):5193.

Selmi A, Vascotto F, Kautz-Neu K, Türeci Ö, Sahin U, von Stebut E, Diken M, Kreiter S. (2016) Uptake of synthetic naked RNA by skin-resident dendritic cells via macropinocytosis allows antigen expression and induction of T-cell responses in mice. Cancer Immunol. Immunother. 65(9):1075-83.

Kranz LM, Diken M, Haas H, Kreiter S, Loquai C, Reuter KC, Meng M, Fritz D, Vascotto F, Hefesha H, Grunwitz C, Vormehr M, Hüsemann Y, Selmi A, Kuhn AN, Buck J, Derhovanessian E, Rae R, Attig S, Diekmann J, Jabulowsky RA, Heesch S, Hassel J, Langguth P, Grabbe S, Huber C, Türeci Ö, Sahin U. (2016) Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy. Nature. 534(7607):396-401.

Kreiter S, Vormehr M, van de Roemer, Niels, Diken M, Löwer M, Diekmann J, Boegel S, Schrörs B, Vascotto F, Castle JC, Tadmor AD, Schoenberger SP, Huber C, Türeci Ö, Sahin U. (2015) Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature.  520:692-696.

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