Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Feb 20;203(2):437-47.
doi: 10.1084/jem.20051775. Epub 2006 Feb 13.

Complete differentiation of CD8+ T cells activated locally within the transplanted liver

Ingo Klein  1 , Ian Nicholas Crispe
Affiliations

Complete differentiation of CD8+ T cells activated locally within the transplanted liver

Ingo Klein et al. J Exp Med. .

Abstract

The transplanted liver elicits systemic tolerance, and the underlying mechanism may also account for the persistence of liver infections, such as malaria and viral hepatitis. These phenomena have led to the hypothesis that antigen presentation within the liver is abortive, leading to T cell tolerance or apoptosis. Here we test this hypothesis in an optimized orthotopic liver transplantation model. In direct contradiction to this model, the liver itself induces full CD8+ T cell activation and differentiation. The effects of microchimerism were neutralized by bone marrow transplantation in the liver donor, and the lack of liver-derived antigen-presenting cells was documented by eight-color flow cytometry and by sensitive functional assays. We conclude that local antigen presentation cannot explain liver tolerance. On the contrary, the liver may be an excellent priming site for naive CD8+ T cells.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Experimental design and histologic results of mouse liver transplantation. (A) Mouse liver transplantation with complete replacement of the recipient's liver by a donor graft was used to limit antigen presentation to the liver. Livers from C57BL/6 mice were transplanted into bm8 mice that are unable to present SIINFEKL peptide on MHC class I. (B) The protocol was refined to eliminate extrahepatic antigen presentation by graft-derived passenger leukocytes. Liver donors were irradiated and reconstituted with recipient-type bone marrow 4 wk before transplantation. 4 wk after transplantation, transgenic OT-I cells were adoptively transferred into transplant recipients that presented the specific peptide only within the transplanted livers. Histological sections of naive B6 control animals (C) and bm8 transplant recipients of livers from radiation bone marrow chimeras (D). Hematoxylin and eosin staining at a magnification of 200, and inset at a magnification of 300 (scale bar, 100 μm). The transplanted livers showed no evidence of rejection.
Figure 2.
Figure 2.
Microchimerism in transplant recipients of liver grafts from untreated B6 and [bm8→B6] radiation bone marrow chimeras. (A) bm8 (CD45.2) recipients of untreated B6.SJL (CD45.1) livers were killed 4 wk after liver transplantation. The frequency and phenotype of donor-derived passenger leukocytes in spleens and peripheral lymph nodes was assessed by eight-color flow cytometry based on the congenic marker CD45.1. (B) Microchimerism in recipients of livers from radiation bone marrow chimeras, in which the bone marrow–derived cells were replaced by bone marrow of the recipient mouse strain (bm8) 4 wk before liver transplantation. Data are representative of two independent experiments with three mice per group. Bottom: average percentage (±SEM) of donor-derived passenger leukocytes as a fraction of the total number of donor-derived CD45.1+ cells (six animals per group). The radiation reduced microchimerism by 90% and abolished the transfer of professional APCs (dendritic cells and B cells).
Figure 3.
Figure 3.
Organ-specific detection of antigen presentation by ex vivo T cell proliferation assay. Antigen presentation in spleens, peripheral and mesenteric lymph nodes, bone marrow, and livers (unfractionated and fractionated into CD45+ and CD45− cells) was assessed independently using an in vitro T cell proliferation assay 4 wk after transplantation. bm8 recipients of liver transplants from [bm8→B6] bone marrow chimeras (hepatic kb expression, open graphs) were compared with B6 recipients of liver grafts from [B6→B6] bone marrow chimeras (systemic kb expression, filled graphs). 4 wk after transplantation, transplant recipients were injected with SIINFEKL peptide, and then cell suspensions from spleens, peripheral and mesenteric lymph nodes, bone marrow, and livers were cocultured with CFSE-stained OT-I cells (CD90.1 background). Organ-specific antigen presentation was determined by dilution of CFSE in OT-I cells. The histograms show OT-I T cells based on their expression of CD90.1. These data demonstrate that the capacity to present the SIINFEKL peptide in the hepatic kb expression group was exclusive to the CD45− and unfractionated intrahepatic cells of the transplanted livers. Data are representative of three independent experiments with two mice per group, and fractionation of liver cells based on CD45 expression was repeated twice with two independently analyzed animals in each group.
Figure 4.
Figure 4.
In vivo expansion of naive OT-I T cells after hepatic and systemic antigen presentation. Naive OT-I cells for adoptive transfer were obtained by magnetic bead depletion, followed by FACS sorting for transgenic T cells with a naive phenotype. (A) Purity and activation status of adoptively transferred OT-I T cells before and after flow cytometric cell sorting. (B) Expansion of adoptively transferred OT-I T cells detected in livers, spleens, and peripheral lymph nodes of transplant recipients. For restricted hepatic kb antigen presentation, livers from [bm8→B6] bone marrow chimeras were transplanted into bm8 recipients. Systemic kb antigen presentation was achieved by transplanting livers from [B6→B6] bone marrow chimeras into B6 recipients. 4 wk after transplantation, 5 × 106 naive OT-I cells (CD90.1 background) were adoptively transferred and the animals received three i.p. injections of either SIINFEKL peptide or PBS (hepatic kb PBS control). 4 d after the initial antigen contact, transplant recipients were killed and the number of OT-I cells was assessed by flow cytometry. Data are representative of three independent experiments with three mice per group, and sorted and unsorted OT-I cells were used for adoptive transfer without significant differences in the extent of prolif-eration. This shows that antigen presentation in the transplanted liver can cause extensive clonal expansion.
Figure 5.
Figure 5.
Kinetics and activation status of intrahepatically activated OT-I T cells. Left column: Absolute cell numbers of adoptively transferred OT-I T cells 2, 4, and 6 d after initial SIINFEKL injection. Transplant recipients with hepatic or systemic capability to present antigen received 5 × 106 OT-I T cells and were injected with SIINFEKL or PBS (hepatic kb PBS control) on days 0, 1, and 2. OT-I cell numbers from livers, spleens, and peripheral lymph nodes were calculated based on flow cy-tometry data and organ cell counts. Right column: Proliferation and activation status of OT-T cells in animals with hepatic and systemic kb antigen presentation demonstrated by dilution of CFSE staining and expression of CD44 on day 4 after initial antigen or PBS injection. The data show that CD8+ T cell activation in the liver causes massive proliferation and seeding of the lymphoid organs. The data are representative of three independent experiments with three mice per group, and error bars indicate SEM.
Figure 6.
Figure 6.
IFN-γ production of OT-I T cells after restimulation with cognate antigen. (A) Liver transplant recipients with restricted intrahepatic or systemic antigen presentation were killed on day 4 after initial antigen injection. Cell isolates from livers, spleens, and lymph nodes were restimulated with SIINFEKL peptide for 6 h and their IFN-γ production was assessed by intracellular cytokine staining. (B) Average percentage of IFN-γ producers among isolated OT-I T cells. These experiments show that priming in liver produces fully functional CD8+ T cells. Data are representative of three independent experiments with three mice per group, and the bars in B represent the mean ± SEM.
Figure 7.
Figure 7.
In vivo CTL challenge with SIINFEKL-pulsed target cells. (A) OT-I cells were adoptively transferred into transplant recipients with restricted hepatic or systemic kb antigen presentation and primed by SIINFEKL injection. 5–6 d after initial antigen encounter, mice were tested for cytotoxic T cells by injection of SIINFEKL-pulsed target cells. Splenocytes from naive B6 animals, stained in a 2-μM CFSE solution (CFSEhigh) and pulsed in a 1-μM SIINFEKL solution, were used as specific target cells. Nonspecific control targets were stained in a 0.2-μM CFSE solution (CFSElow) and pulsed with the influenza PA peptide. Specific and nonspecific target cells were mixed in a 1:1 ratio, and a total of 2 × 107 cells was injected i.v. into transplant recipients (A, preinjection). 4 h after the injection of target cells, transplant recipients were killed and cell suspensions from livers, spleens, and peripheral lymph nodes were analyzed for CFSE+ cells by flow cytometry (A, right). (B) Cytotoxicity was determined by the increased ratio of specific versus nonspecific target cells (see Results and Materials and methods for details). Cytotoxic activity of endogenous SIINFEKL-specific cells was excluded in transplant recipients by three injections of cognate antigen in the absence of previous adoptive OT-I transfer (endogenous SIINFEKL control). (C) Percentage of specific cytotoxicity in transplant recipients. These experiments show that antigen presented in the transplanted liver can prime CTLs. Data are representative of three independent experiments with three mice per group, and the bars in C represent the average ± SEM.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Rehermann, B., and M. Nascimbeni. 2005. Immunology of hepatitis B virus and hepatitis C virus infection. Nat. Rev. Immunol. 5:215–229. - PubMed
    1. Good, M.F. 1995. Development of immunity to malaria may not be an entirely active process. Parasite Immunol. 17:55–59. - PubMed
    1. Calne, R., and H. Davies. 1994. Organ graft tolerance: the liver effect. Lancet. 343:67–68. - PubMed
    1. Gassel, H.J., I.V. Hutchinson, R. Engemann, and P.J. Morris. 1992. The role of T suppressor cells in the maintenance of spontaneously accepted orthotopic rat liver allografts. Transplantation. 54:1048–1053. - PubMed
    1. Farges, O., P.J. Morris, and M.J. Dallman. 1994. Spontaneous acceptance of liver allografts in the rat. Analysis of the immune response. Transplantation. 57:171–177. - PubMed

Publication types

MeSH terms

Substances