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Visualizing the innate and adaptive immune responses underlying allograft rejection by two-photon microscopy


Transplant rejection involves a coordinated attack of the innate and the adaptive immune systems of the host. To investigate this dynamic process and the contributions of both donor and host cells, we developed an ear skin graft model suitable for intravital imaging. We found that donor dermal dendritic cells (DCs) migrated rapidly from the graft and were replaced by host CD11b+ mononuclear cells. The infiltrating host cells captured donor antigen, reached the draining lymph node and cross-primed graft-reactive CD8+ T cells. Furthermore, we defined the mechanisms by which host T cells target graft cells. We found that primed T cells entered the graft from the surrounding tissue and localized selectively at the dermis-epidermis junction. Later, CD8+ T cells disseminated throughout the graft and many became arrested. These results provide insights into the antigen presentation pathway and the stepwise progression of CD8+ T cell activity, thereby offering a framework for evaluating how immunotherapy might abrogate the key steps in allograft rejection.

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Figure 1: Donor Langerhans cells but not dermal DCs persist in the graft.
Figure 2: Spatiotemporal pattern of graft infiltration by host cells.
Figure 3: Recipient graft-infiltrating cells reach the dLN and cross-prime CD8+ T cells.
Figure 4: Priming and graft localization of CD8+ T cells in the early phase of rejection.
Figure 5: CD8+ T cell dynamics and cytotoxic activity in the graft.

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  1. Auchincloss, H. Jr. & Sultan, H. Antigen processing and presentation in transplantation. Curr. Opin. Immunol. 8, 681–687 (1996).

    Article  CAS  Google Scholar 

  2. Rosenberg, A.S. & Singer, A. Cellular basis of skin allograft rejection: an in vivo model of immune-mediated tissue destruction. Annu. Rev. Immunol. 10, 333–360 (1992).

    Article  CAS  Google Scholar 

  3. Ingulli, E. Mechanism of cellular rejection in transplantation. Pediatr. Nephrol. 25, 61–74 (2010).

    Article  Google Scholar 

  4. Talmage, D.W., Dart, G., Radovich, J. & Lafferty, K.J. Activation of transplant immunity: effect of donor leukocytes on thyroid allograft rejection. Science 191, 385–388 (1976).

    Article  CAS  Google Scholar 

  5. Richards, D.M. et al. Indirect minor histocompatibility antigen presentation by allograft recipient cells in the draining lymph node leads to the activation and clonal expansion of CD4+ T cells that cause obliterative airways disease. J. Immunol. 172, 3469–3479 (2004).

    Article  CAS  Google Scholar 

  6. Ochando, J.C., Krieger, N.R. & Bromberg, J.S. Direct versus indirect allorecognition: Visualization of dendritic cell distribution and interactions during rejection and tolerization. Am. J. Transplant. 6, 2488–2496 (2006).

    Article  CAS  Google Scholar 

  7. Larsen, C.P., Austyn, J.M. & Morris, P.J. The role of graft-derived dendritic leukocytes in the rejection of vascularized organ allograft. Recent findings on the migration and function of dendritic leukocytes after transplantation. Ann. Surg. 212, 308–315 (1990).

    Article  CAS  Google Scholar 

  8. Valujskikh, A., Hartig, C. & Heeger, P.S. Indirectly primed CD8+ T cells are a prominent component of the allogeneic T-cell repertoire after skin graft rejection in mice. Transplantation 71, 418–421 (2001).

    Article  CAS  Google Scholar 

  9. Garrod, K.R. et al. NK cell patrolling and elimination of donor-derived dendritic cells favor indirect alloreactivity. J. Immunol. 184, 2329–2336 (2010).

    Article  CAS  Google Scholar 

  10. Lakkis, F.G., Arakelov, A., Konieczny, B.T. & Inoue, Y. Immunologic 'ignorance' of vascularized organ transplants in the absence of secondary lymphoid tissue. Nat. Med. 6, 686–688 (2000).

    Article  CAS  Google Scholar 

  11. Zhou, P. et al. Secondary lymphoid organs are important but not absolutely required for allograft responses. Am. J. Transplant. 3, 259–266 (2003).

    Article  Google Scholar 

  12. Barker, C.F. & Billingham, R.E. The role of afferent lymphatics in the rejection of skin homografts. J. Exp. Med. 128, 197–221 (1968).

    Article  CAS  Google Scholar 

  13. Wang, J. et al. Donor lymphoid organs are a major site of alloreactive T-cell priming following intestinal transplantation. Am. J. Transplant. 6, 2563–2571 (2006).

    Article  CAS  Google Scholar 

  14. Schenk, A.D., Nozaki, T., Rabant, M., Valujskikh, A. & Fairchild, R.L. Donor-reactive CD8 memory T cells infiltrate cardiac allografts within 24-h post-transplant in naive recipients. Am. J. Transplant. 8, 1652–1661 (2008).

    Article  CAS  Google Scholar 

  15. Kreisel, D. et al. Non-hematopoietic allograft cells directly activate CD8+ T cells and trigger acute rejection: an alternative mechanism of allorecognition. Nat. Med. 8, 233–239 (2002).

    Article  CAS  Google Scholar 

  16. Gelman, A.E. et al. Cutting edge: Acute lung allograft rejection is independent of secondary lymphoid organs. J. Immunol. 182, 3969–3973 (2009).

    Article  CAS  Google Scholar 

  17. Rocha, P.N., Plumb, T.J., Crowley, S.D. & Coffman, T.M. Effector mechanisms in transplant rejection. Immunol. Rev. 196, 51–64 (2003).

    Article  CAS  Google Scholar 

  18. Strid, J. et al. Acute upregulation of an NKG2D ligand promotes rapid reorganization of a local immune compartment with pleiotropic effects on carcinogenesis. Nat. Immunol. 9, 146–154 (2008).

    Article  CAS  Google Scholar 

  19. Nishibu, A. et al. Behavioral responses of epidermal Langerhans cells in situ to local pathological stimuli. J. Invest. Dermatol. 126, 787–796 (2006).

    Article  CAS  Google Scholar 

  20. Peters, N.C. et al. In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science 321, 970–974 (2008).

    Article  CAS  Google Scholar 

  21. Chtanova, T. et al. Dynamics of neutrophil migration in lymph nodes during infection. Immunity 29, 487–496 (2008).

    Article  CAS  Google Scholar 

  22. Matsuo, S., Kurisaki, A., Sugino, H., Hashimoto, I. & Nakanishi, H. Analysis of skin graft survival using green fluorescent protein transgenic mice. J. Med. Invest. 54, 267–275 (2007).

    Article  Google Scholar 

  23. Ehst, B.D., Ingulli, E. & Jenkins, M.K. Development of a novel transgenic mouse for the study of interactions between CD4 and CD8 T cells during graft rejection. Am. J. Transplant. 3, 1355–1362 (2003).

    Article  CAS  Google Scholar 

  24. Konjufca, V. & Miller, M.J. Two-photon microscopy of host-pathogen interactions: acquiring a dynamic picture of infection in vivo. Cell. Microbiol. 11, 551–559 (2009).

    Article  CAS  Google Scholar 

  25. Ng, L.G., Mrass, P., Kinjyo, I., Reiner, S.L. & Weninger, W. Two-photon imaging of effector T-cell behavior: lessons from a tumor model. Immunol. Rev. 221, 147–162 (2008).

    Article  CAS  Google Scholar 

  26. Kissenpfennig, A. et al. Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells. Immunity 22, 643–654 (2005).

    Article  CAS  Google Scholar 

  27. Sen, D., Forrest, L., Kepler, T.B., Parker, I. & Cahalan, M.D. Selective and site-specific mobilization of dermal dendritic cells and Langerhans cells by TH1- and TH2-polarizing adjuvants. Proc. Natl. Acad. Sci. USA 107, 8334–8339 (2010).

    Article  CAS  Google Scholar 

  28. Wang, L. et al. Langerin expressing cells promote skin immune responses under defined conditions. J. Immunol. 180, 4722–4727 (2008).

    Article  CAS  Google Scholar 

  29. Obhrai, J.S. et al. Langerhans cells are not required for efficient skin graft rejection. J. Invest. Dermatol. 128, 1950–1955 (2008).

    Article  CAS  Google Scholar 

  30. Beil, W.J., Meinardus-Hager, G., Neugebauer, D.C. & Sorg, C. Differences in the onset of the inflammatory response to cutaneous leishmaniasis in resistant and susceptible mice. J. Leukoc. Biol. 52, 135–142 (1992).

    Article  CAS  Google Scholar 

  31. Léon, B., Lopez-Bravo, M. & Ardavin, C. Monocyte-derived dendritic cells formed at the infection site control the induction of protective T helper 1 responses against Leishmania. Immunity 26, 519–531 (2007).

    Article  Google Scholar 

  32. Robben, P.M., LaRegina, M., Kuziel, W.A. & Sibley, L.D. Recruitment of Gr-1+ monocytes is essential for control of acute toxoplasmosis. J. Exp. Med. 201, 1761–1769 (2005).

    Article  CAS  Google Scholar 

  33. Benichou, G., Valujskikh, A. & Heeger, P.S. Contributions of direct and indirect T cell alloreactivity during allograft rejection in mice. J. Immunol. 162, 352–358 (1999).

    CAS  PubMed  Google Scholar 

  34. Le Borgne, M. et al. Dendritic cells rapidly recruited into epithelial tissues via CCR6/CCL20 are responsible for CD8+ T cell crosspriming in vivo. Immunity 24, 191–201 (2006).

    Article  CAS  Google Scholar 

  35. Valujskikh, A., Lantz, O., Celli, S., Matzinger, P. & Heeger, P.S. Cross-primed CD8+ T cells mediate graft rejection via a distinct effector pathway. Nat. Immunol. 3, 844–851 (2002).

    Article  CAS  Google Scholar 

  36. Rosenberg, A.S. & Singer, A. Evidence that the effector mechanism of skin allograft rejection is antigen-specific. Proc. Natl. Acad. Sci. USA 85, 7739–7742 (1988).

    Article  CAS  Google Scholar 

  37. Breart, B., Lemaitre, F., Celli, S. & Bousso, P. Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice. J. Clin. Invest. 118, 1390–1397 (2008).

    Article  CAS  Google Scholar 

  38. Boissonnas, A., Fetler, L., Zeelenberg, I.S., Hugues, S. & Amigorena, S. In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor. J. Exp. Med. 204, 345–356 (2007).

    Article  CAS  Google Scholar 

  39. Van den Broeck, W., Derore, A. & Simoens, P. Anatomy and nomenclature of murine lymph nodes: Descriptive study and nomenclatory standardization in BALB/cAnNCrl mice. J. Immunol. Methods 312, 12–19 (2006).

    Article  CAS  Google Scholar 

  40. Celli, S., Garcia, Z. & Bousso, P. CD4 T cells integrate signals delivered during successive DC encounters in vivo. J. Exp. Med. 202, 1271–1278 (2005).

    Article  CAS  Google Scholar 

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We wish to thank H. Saklani and C. Auriau (Institut Pasteur) for providing B2m−/− mOVA mice and E. Robey and the members of the Bousso laboratory for comments on the manuscript. This work was supported by INSERM, Institut Pasteur and a Marie Curie Excellence grant.

Author information

Authors and Affiliations



S.C. designed and carried out the experiments, analyzed the data and wrote the manuscript; M.L.A. developed crucial reagents and participated in experimental design; and P.B. designed the experiments, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Philippe Bousso.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 1093 kb)

Supplementary Video 1

Mobility of donor dendritic cells in the graft (MOV 2340 kb)

Supplementary Video 2

Donor DCs rapidly die in the draining lymph node (MOV 2012 kb)

Supplementary Video 3

The motility of graft-infiltrating host cells changes over time (MOV 1147 kb)

Supplementary Video 4

Infiltration of the epidermis in the presence of antigenic mismatch (MOV 2506 kb)

Supplementary Video 5

Recipient graft-infiltrating cells are visualized following retransplant (MOV 1091 kb)

Supplementary Video 6

Recipient graft-infiltrating cells can reach the draining lymph node (MOV 2089 kb)

Supplementary Video 7

Skin allografts are efficiently revascularized on day 5 (MOV 3659 kb)

Supplementary Video 8

CD8+ T cells accumulate in the recipient tissue around the graft (MOV 3789 kb)

Supplementary Video 9

CD4+ T cells accumulate in the recipient tissue around the graft (MOV 660 kb)

Supplementary Video 10

CD4+ T cells localize at the dermis/epidermis junction as they enter the graft (MOV 931 kb)

Supplementary Video 11

CD8+ T cells colocalize with propidium iodide positive cells in the allograft (MOV 3328 kb)

Supplementary Video 12

CD8+ T cells colocalize with propidium iodide positive cells in the allograft (MOV 4226 kb)

Supplementary Video 13

CD8+ T cells colocalize with propidium iodide positive cells in the allograft (MOV 686 kb)

Supplementary Video 14

CD8+ T cells colocalize with propidium iodide positive cells in the allograft (MOV 1068 kb)

Supplementary Video 15

CD8+ T cells displayed reduced motility and increased confinement in the allograft (MOV 3723 kb)

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Celli, S., Albert, M. & Bousso, P. Visualizing the innate and adaptive immune responses underlying allograft rejection by two-photon microscopy. Nat Med 17, 744–749 (2011).

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