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

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.