Journal of Molecular Medicine

, Volume 96, Issue 3–4, pp 349–360 | Cite as

Imaging of cytotoxic antiviral immunity while considering the 3R principle of animal research

  • Lucas Otto
  • Gennadiy Zelinskyy
  • Marc Schuster
  • Ulf DittmerEmail author
  • Matthias GunzerEmail author
Original Article


Adoptive cell transfer approaches for antigen-specific CD8+ T cells are used widely to study their effector potential during infections or cancer. However, contemporary methodological adaptations regarding transferred cell numbers, advanced imaging, and the 3R principle of animal research have been largely omitted. Here, we introduce an improved cell transfer method that reduces the number of donor animals substantially and fulfills the requirements for intravital imaging under physiological conditions. For this, we analyzed the well-established Friend retrovirus (FV) mouse model. Donor mice that expressed a FV-specific T cell receptor (TCRtg) and the fluorescent protein tdTomato were used as source of antigen-specific CD8+ T cells. Only a few drops of peripheral blood were sufficient to isolate ~ 150,000 naive reporter cells from which 1000 were adoptively transferred into recently FV-infected recipients. The cells became activated and functional and expanded strongly in the spleen and bone marrow within 10 days post infection. Transferred CD8+ T cells participated in the antiviral host response within a natural range and developed an effector phenotype indistinguishable from endogenous effector CD8+ T cells. Additionally, the generated reporter cell frequency allowed single cell visualization and tracking of a physiological antiretroviral CD8+ T cell response by intravital two-photon microscopy. Highly reproducible results were obtained in independent experiments by reusing the same donors repetitively for multiple transfers. Our approach allows a strong reduction of experimental animals required for studies on antigen-specific CD8+ T cell function and should be applicable to other transfer models.

Key messages

  • TCRtg CD8+ T cells are obtained repetitively from the blood samples of single donors.

  • One thousand transferred TCRtg CD8+ T cells get activated, are functional, and proliferate.

  • Several adoptive cell transfers from the same donor show reproducible results.

  • One thousand transferred cells take part in the FV immune response without modifying it.

  • Use of fluorescent transfer cells allows in vivo imaging and single cell tracking.


Antigen-specific cytotoxic CD8+ T cells (CTL) Adoptive cell transfer Repetitive donor mouse usage Intravital imaging Retroviruses 3R principle 



The Imaging Center Essen (IMCES) is acknowledged for expert technical support in imaging experiments.

Author contribution

L.O. performed all experiments. G.Z. and M.S. provided essential support. L.O. and M.G. wrote the manuscript with the help of G.Z. and U.D. M.G. and U.D. conceived of and supervised the study.

Funding information

This work was supported by a grant to M.G., U.D., and G.Z. from the Deutsche Forschungsgemeinschaft (DFG, TRR60, Projects B4 and B8), and the European Union (H2020, Multimot) to M.G. L.O. was trained in the DFG-funded RTG 1949.

Compliance with ethical standards

Animal experiments were conducted under strict consent with the German regulations of the Society for Laboratory Animal Science (GV-SOLAS) and the European Health Law of the Federation of Laboratory Animal Science Associations (FELASA). North Rhine-Westphalia State Agency for Nature, Environment and Consumer Protection (LANUV) approved all experiments and protocols.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute for Experimental Immunology and Imaging, University Hospital EssenUniversity Duisburg-EssenEssenGermany
  2. 2.Institute for Virology, University Hospital EssenUniversity Duisburg-EssenEssenGermany

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