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.
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.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Halle S, Keyser KA, Stahl FR, Busche A, Marquardt A, Zheng X, Galla M, Heissmeyer V, Heller K, Boelter J, Wagner K, Bischoff Y, Martens R, Braun A, Werth K, Uvarovskii A, Kempf H, Meyer-Hermann M, Arens R, Kremer M, Sutter G, Messerle M, Förster R (2016) In vivo killing capacity of cytotoxic T cells is limited and involves dynamic interactions and T cell cooperativity. Immunity 44:233–245
Schmitt A, Tonn T, Busch DH, Grigoleit GU, Einsele H, Odendahl M, Germeroth L, Ringhoffer M, Ringhoffer S, Wiesneth M, Greiner J, Michel D, Mertens T, Rojewski M, Marx M, von Harsdorf S, Döhner H, Seifried E, Bunjes D, Schmitt M (2011) Adoptive transfer and selective reconstitution of streptamer-selected cytomegalovirus-specific CD8+ T cells leads to virus clearance in patients after allogeneic peripheral blood stem cell transplantation. Transfusion 51:591–599
Stemberger C, Huster KM, Koffler M, Anderl F, Schiemann M, Wagner H, Busch DH (2007) A single naive CD8+ T cell precursor can develop into diverse effector and memory subsets. Immunity 27:985–997
Kamphorst AO, Wieland A, Nasti T, Yang S, Zhang R, Barber DL, Konieczny BT, Daugherty CZ, Koenig L, Yu K, Sica GL, Sharpe AH, Freeman GJ, Blazar BR, Turka LA, Owonikoko TK, Pillai RN, Ramalingam SS, Araki K, Ahmed R (2017) Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent. Science 355:1423–1427
Dittmer U, He H, Messer RJ, Schimmer S, Olbrich AR, Ohlen C, Greenberg PD, Stromnes IM, Iwashiro M, Sakaguchi S et al (2004) Functional impairment of CD8(+) T cells by regulatory T cells during persistent retroviral infection. Immunity 20:293–303
Hukelmann JL, Anderson KE, Sinclair LV, Grzes KM, Murillo AB, Hawkins PT, Stephens LR, Lamond AI, Cantrell DA (2016) The cytotoxic T cell proteome and its shaping by the kinase mTOR. Nat Immunol 17:104–112
Barchet W, Oehen S, Klenerman P, Wodarz D, Bocharov G, Lloyd AL, Nowak MA, Hengartner H, Zinkernagel RM, Ehl S (2000) Direct quantitation of rapid elimination of viral antigen-positive lymphocytes by antiviral CD8(+) T cells in vivo. Eur J Immunol 30:1356–1363
Badovinac VP, Haring JS, Harty JT (2007) Initial T cell receptor transgenic cell precursor frequency dictates critical aspects of the CD8(+) T cell response to infection. Immunity 26:827–841
Marzo AL, Klonowski KD, Le Bon A, Borrow P, Tough DF, Lefrancois L (2005) Initial T cell frequency dictates memory CD8+ T cell lineage commitment. Nat Immunol 6:793–799
Blattman JN, Antia R, Sourdive DJ, Wang X, Kaech SM, Murali-Krishna K, Altman JD, Ahmed R (2002) Estimating the precursor frequency of naive antigen-specific CD8 T cells. J Exp Med 195:657–664
Stock AT, Mueller SN, Kleinert LM, Heath WR, Carbone FR, Jones CM (2007) Optimization of TCR transgenic T cells for in vivo tracking of immune responses. Immunol Cell Biol 85:394–396
Myers L, Hasenkrug KJ (2009) Retroviral immunology: lessons from a mouse model. Immunol Res 43:160–166
Hasenkrug KJ, Dittmer U (2007) Immune control and prevention of chronic Friend retrovirus infection. Front Biosci 12:1544–1551
Chen W, Qin H, Chesebro B, Cheever MA (1996) Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors. J Virol 70:7773–7782
Ohlen C, Kalos M, Cheng LE, Shur AC, Hong DJ, Carson BD, Kokot NC, Lerner CG, Sather BD, Huseby ES et al (2002) CD8(+) T cell tolerance to a tumor-associated antigen is maintained at the level of expansion rather than effector function. J Exp Med 195:1407–1418
Germain RN, Robey EA, Cahalan MD (2012) A decade of imaging cellular motility and interaction dynamics in the immune system. Science 336:1676–1681
Mempel TR, Henrickson SE, von Andrian UH (2004) T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 427:154–159
Breart B, Lemaitre F, Celli S, Bousso P (2008) Two-photon imaging of intratumoral CD8 T cell cytotoxic activity during adoptive T cell therapy in mice. J Clin Invest 118:1390–1397
Eickhoff S, Brewitz A, Gerner MY, Klauschen F, Komander K, Hemmi H, Garbi N, Kaisho T, Germain RN, Kastenmuller W (2015) Robust anti-viral immunity requires multiple distinct T cell-dendritic cell interactions. Cell 162:1322–1337
Liu Z, Gerner MY, Van PN, Levine AG, Rudensky AY, Germain RN (2015) Immune homeostasis enforced by co-localized effector and regulatory T cells. Nature 528:225–230
Deguine J, Breart B, Lemaitre F, Di Santo JP, Bousso P (2010) Intravital imaging reveals distinct dynamics for natural killer and CD8(+) T cells during tumor regression. Immunity 33:632–644
Beuneu H, Lemaitre F, Deguine J, Moreau HD, Bouvier I, Garcia Z, Albert ML, Bousso P (2010) Visualizing the functional diversification of CD8(+) T cell responses in lymph nodes. Immunity 33:412–423
Scholer A, Hugues S, Boissonnas A, Fetler L, Amigorena S (2008) Intercellular adhesion molecule-1-dependent stable interactions between T cells and dendritic cells determine CD8+ T cell memory. Immunity 28:258–270
Gunzer M, Weishaupt C, Hillmer A, Basoglu Y, Friedl P, Dittmar KE, Kolanus W, Varga G, Grabbe S (2004) A spectrum of biophysical interaction modes between T cells and different antigen presenting cells during priming in 3-D collagen and in vivo. Blood 104:2801–2809
Russell WMS, Burch RL (1959) The principles of humane experimental technique. Methuen, London
Lahl K, Loddenkemper C, Drouin C, Freyer J, Arnason J, Eberl G, Hamann A, Wagner H, Huehn J, Sparwasser T (2007) Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease. J Exp Med 204:57–63
Zhang DJ, Wang Q, Wei J, Baimukanova G, Buchholz F, Stewart AF, Mao X, Killeen N (2005) Selective expression of the Cre recombinase in late-stage thymocytes using the distal promoter of the Lck gene. J Immunol 174:6725–6731
Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng H (2010) A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13:133–140
Lilly F, Steeves RA (1973) B-tropic Friend virus: a host-range pseudotype of spleen focus-forming virus (SFFV). Virology 55:363–370
Robertson SJ, Ammann CG, Messer RJ, Carmody AB, Myers L, Dittmer U, Nair S, Gerlach N, Evans LH, Cafruny WA, Hasenkrug KJ (2008) Suppression of acute anti-friend virus CD8+ T-cell responses by coinfection with lactate dehydrogenase-elevating virus. J Virol 82:408–418
Grupillo M, Lakomy R, Geng X, Styche A, Rudert WA, Trucco M, Fan Y (2011) An improved intracellular staining protocol for efficient detection of nuclear proteins in YFP-expressing cells. BioTechniques 51:417–420
Köhler A, De Filippo K, Hasenberg M, van den Brandt C, Nye E, Hosking MP, Lane TE, Männ L, Ransohoff RM, Hauser AE et al (2011) G-CSF mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands. Blood 117:4349–4357
Robertson MN, Miyazawa M, Mori S, Caughey B, Evans LH, Hayes SF, Chesebro B (1991) Production of monoclonal antibodies reactive with a denatured form of the Friend murine leukemia virus gp70 envelope protein: use in a focal infectivity assay, immunohistochemical studies, electron microscopy and western blotting. J Virol Methods 34:255–271
Berke G (1994) The binding and lysis of target cells by cytotoxic lymphocytes: molecular and cellular aspects. Annu Rev Immunol 12:735–773
Betts MR, Brenchley JM, Price DA, De Rosa SC, Douek DC, Roederer M, Koup RA (2003) Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281:65–78
Zelinskyy G, Dietze KK, Husecken YP, Schimmer S, Nair S, Werner T, Gibbert K, Kershaw O, Gruber AD, Sparwasser T, Dittmer U (2009) The regulatory T-cell response during acute retroviral infection is locally defined and controls the magnitude and duration of the virus-specific cytotoxic T-cell response. Blood 114:3199–3207
Mazo IB, Honczarenko M, Leung H, Cavanagh LL, Bonasio R, Weninger W, Engelke K, Xia L, McEver RP, Koni PA et al (2005) Bone marrow is a major reservoir and site of recruitment for central memory CD8+ T cells. Immunity 22:259–270
Miller MJ, Hejazi AS, Wei SH, Cahalan MD, Parker I (2004) T cell repertoire scanning is promoted by dynamic dendritic cell behavior and random T cell motility in the lymph node. Proc Natl Acad Sci U S A 101:998–1003
Gunzer M, Schäfer A, Borgmann S, Grabbe S, Zänker KS, Bröcker E-B, Kämpgen E, Friedl P (2000) Antigen presentation in extracellular matrix: interactions of T cells with dendritic cells are dynamic, short lived, and sequential. Immunity 13:323–332
Yoon H, Kim TS, Braciale TJ (2010) The cell cycle time of CD8+ T cells responding in vivo is controlled by the type of antigenic stimulus. PLoS One 5:e15423. https://doi.org/10.1371/journal.pone.0015423
Newell EW, Sigal N, Bendall SC, Nolan GP, Davis MM (2012) Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes. Immunity 36:142–152
Giovanoli S, Engler H, Engler A, Richetto J, Voget M, Willi R, Winter C, Riva MA, Mortensen PB, Feldon J, Schedlowski M, Meyer U (2013) Stress in puberty unmasks latent neuropathological consequences of prenatal immune activation in mice. Science 339:1095–1099
The Imaging Center Essen (IMCES) is acknowledged for expert technical support in imaging experiments.
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.
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.
Electronic supplementary material
(DOCX 16 kb)
(DOCX 15 kb)
(MPG 7950 kb)
(GIF 212 kb)
(GIF 81 kb)
(GIF 240 kb)
(GIF 588 kb)
(GIF 438 kb)
(GIF 316 kb)
About this article
Cite this article
Otto, L., Zelinskyy, G., Schuster, M. et al. Imaging of cytotoxic antiviral immunity while considering the 3R principle of animal research. J Mol Med 96, 349–360 (2018). https://doi.org/10.1007/s00109-018-1628-7
- Antigen-specific cytotoxic CD8+ T cells (CTL)
- Adoptive cell transfer
- Repetitive donor mouse usage
- Intravital imaging
- 3R principle