View larger version (42K): [in this window] [in a new window]   Figure 2. Microchimerism in transplant recipients of liver grafts from untreated B6 and [bm8B6] 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).

View larger version (35K): [in this window] [in a new window]   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 [bm8B6] bone marrow chimeras (hepatic kb expression, open graphs) were compared with B6 recipients of liver grafts from [B6B6] 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.

View larger version (48K): [in this window] [in a new window]   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 [bm8B6] bone marrow chimeras were transplanted into bm8 recipients. Systemic kb antigen presentation was achieved by transplanting livers from [B6B6] bone marrow chimeras into B6 recipients. 4 wk after transplantation, 5 x 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.

View larger version (41K): [in this window] [in a new window]   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 x 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.

CD8+ T cells acquire full effector functions when primed in the liver
Because naive CD8⁺ T cells responded to intrahepatic antigen presentation by activation and proliferation, we were interested in the functional potential of these cells. OT-I cells from liver grafts, spleens, and lymph nodes were isolated on days 2 and 4 after antigen encounter, and their ability to produce IFN- was tested by intracellular cytokine staining after a 6-h restimulation period. Restricted hepatic antigen presentation had no negative effect on IFN- production, which was the same as that observed in mice with systemic presentation (Fig. 6 A). In the absence of antigen restimulation (media without added SIINFEKL peptide), no significant cytokine production was detectable at the end of the culture period. OT-I cells that were primed within liver grafts readily produced IFN- upon restimulation, as early as 2 d after antigen contact. During the course of the immune response, IFN-–producing OT-I cells initially recirculated into peripheral lymphatic organs and later accumulated in the livers of both groups, resulting in a higher percentage compared with spleens and lymph nodes on day 4 of the immune response (Fig. 4 B). Despite this higher percentage in the liver, the fraction of OT-I cells that responded to peptide restimulation by IFN- production was not significantly different between the liver and the lymphatic organs (Fig. 6 B; liver, P = 0.77; spleen, P = 0.52; lymph nodes, P = 0.71). This indicates that the liver environment does not negatively influence the ability of CD8⁺ T cells to produce IFN-. In PBS-injected transplant recipients, no cytokine production was observed 6 h after restimulation. Also, the percentage of cytokine-producing OT-I cells as well as the total OT-I cell number in transplanted animals was similar to that observed in nontransplanted B6 mice after OT-I transfer and peptide injection. This indicated that the transplantation technique did not affect the quality of the immune response as determined by cytokine production (not depicted).

View larger version (37K): [in this window] [in a new window]   Figure 6. IFN- 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- production was assessed by intracellular cytokine staining. (B) Average percentage of IFN- 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.

View larger version (48K): [in this window] [in a new window]   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 x 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.

	DISCUSSION

Top Abstract RESULTS DISCUSSION MATERIALS AND METHODS References

In this work, this hypothesis was tested using a novel approach. Antigen was acutely introduced and its presentation was restricted to resident cells of a previously transplanted liver graft. Surprisingly, local intrahepatic antigen presentation in our model was sufficient for the full activation and differentiation of naive CD8⁺ T cells without the need for initial priming in lymphatic organs.

It is generally agreed that CD4⁺ T cell responses are dependent on antigen presentation and costimulatory signals of mature dendritic cells within regional lymph nodes, and that priming does not occur outside lymphatic organs (30). In the case of initial priming of CD8⁺ lymphocytes, the absolute need for costimulation is less well established. Full activation and differentiation was observed in CD28-deficient mice that were infected with lymphocytic choriomeningitis virus (31), and the T cell–mediated destruction of pancreatic ß cells was unaffected in CD28-deficient nonobese diabetic mice (32). In vitro, full activation and differentiation of transgenic CD8⁺ T cells has been induced by peptide-loaded tetramers, despite the lack of costimulation (33). From these studies it would be predicted that the special costimulatory properties of dendritic cells are optional for CD8⁺ T cells. The current work, in which we show that priming and full activation of naive CD8⁺ T cells can occur independent of lymphatic organs, is consistent with this view of CD8⁺ T cell priming. The complete, acute activation and differentiation of naive CD8⁺ T cells was demonstrated by specific cytotoxicity until day 6 after initial peptide activation. This argues against a defective initial activation of naive CD8 T⁺ cells as the exclusive reason of liver-mediated CD8⁺ T cell tolerance. Because this experimental model depends on the intrahepatic expression of an MHC class I restriction element, and the antigen is a class I–restricted peptide, the model excludes the participation of CD4⁺ T helper cells, which have been shown to be essential for the long-term survival of memory cells. Without help, memory CD8⁺ T cell survival is impaired; therefore, we do not draw the conclusion that the fully activated CD8⁺ T cells would be likely to differentiate into memory cells.

In the context of the liver transplantation model, it is appropriate to consider whether the observed CD8⁺ T cell activation could be occurring in neo-lymphatic tissue, induced in the liver grafts by chronic inflammation, due to low-grade rejection. Liver transplants in mice are generally accepted by the recipient without the need for immunosuppression and result in donor-specific tolerance (34). In our model, stable graft acceptance and tolerance induction toward the B6 liver parenchyma was observed by 4 wk after liver transplantation. Adoptive transfer of B6 lymphocytes, either in the form of OT-I cells in the proliferation experiments or as B6 splenocytes in the in vivo cytotoxicity assays, were accepted without rejection, consistent with the induction of full transplantation tolerance in the first 4 wk after transplantation of the chimeric liver graft. In contrast to this, adoptive transfer of B6 cells into either nontransplanted bm8 mice as well as bm8 recipients of [bm8bm8] bone marrow chimeric livers resulted in rejection and complete disappearance of these cells within the first 2 d after transfer (not depicted). However, there was no histological evidence of lymphocyte infiltration or chronic rejection detectable in the liver transplants 4 wk after transplantation (Fig. 1 and Fig. S1). Other potential causes of liver inflammation, such as infection with pathogens (in particular mouse hepatitis virus), were not observed in our mouse colonies for the entire time of the experiments.

Transplantation of whole organs is accompanied by the transfer of passenger leukocytes from the organ donor. These donor-derived cells migrate out of the transplant into the recipient and relocate to lymphoid and nonlymphoid organs, a phenomenon known as microchimerism (35, 36). Consequently, in our model, passenger leukocytes in transplant recipients were a potential source of antigen presentation outside the liver graft. To address this concern, we transplanted the livers from radiation bone marrow chimeras, in which the bone marrow–derived cells were replaced by bone marrow of the recipient mouse strain 4 wk before liver transplantation (Fig. 1 B). This resulted in the complete disappearance of the donor-derived APCs from the recipient's spleens, peripheral lymph nodes, and liver grafts as detected by flow cytometry (Fig. 2 and Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20051775/DC1). To confirm the restriction of antigen presentation to the liver grafts in a sensitive functional assay, we separated hepatic antigen presentation from lymphatic antigen presentation in an ex vivo T cell proliferation experiment. Constituent cells from livers, peripheral and mesenteric lymph nodes, spleens, and bone marrow were evaluated independently for their ability to activate antigen-specific T cells and promote their proliferation. Indeed, this demonstrated that by using bone marrow chimeras as liver donors, microchimerism of effective APCs was eliminated and antigen presentation was restricted to non-bone marrow–derived cells of the liver itself (Fig. 3).

However, the immune response in vivo was not restricted to the transplanted organ. Instead, OT-I T cells recirculated systemically and were found in lymphatic tissues of the recipient early in the immune response. At various time points during the immune response, OT-I cells isolated from spleens, lymph nodes, and livers showed a similar activated phenotype and proliferation status, and resembled each other in their ability to produce IFN- upon restimulation. This suggests constant recirculation between the antigen expressing liver and the lymphatic tissues during the ongoing immune response. The actual site of expansion was not addressed by our experiments, and it is quite conceivable that mitotic cell divisions and/or T cell maturation of OT-I cells took place in lymphatic tissues as well as in the liver. However, the activation and proliferation of CD8⁺ T cells in our model was clearly initiated by antigen presentation in the liver. Surprisingly, there was no significant difference in the magnitude of T cell expansion when antigen was presented systemically or restricted to the liver. Our data indicate that the capacity of the liver to present antigen is sufficient to promote activation and proliferation of a large number of T cells. Several physiologic aspects of the liver contribute to this. The hepatic environment, unlike that of other parenchymal organs, promotes the interaction of naive lymphocytes with potential tissue APCs. The liver is perfused by 25% of the cardiac output, which results in constant exposure of circulating T cells with presented antigen. A unique combination of narrow hepatic sinuses with a fenestrated endothelium and a lack of basement membrane, together with a low velocity blood flow, allow perpetual contact of circulating lymphocytes with potential APCs in the sinusoids and the subendothelial space of Disse (12, 14). In addition, exposure to endotoxins and other bacterial products derived from the intestine provide a constant source of "endogenous" immunological stimulation that results in up-regulation of adhesion molecules on Kupffer cells and LSECs (37).

T cell activation in this special environment has been shown previously. However, the final result of hepatic T cell activation was often premature activation-induced cell death, defective activation and tolerance induction in CD8⁺ T cells, or the formation of a regulatory cell type in CD4⁺ T cells (17, 38). These observations led to the hypothesis that immunological tolerance might be established in the liver by local antigen presentation and implicated a special role in this process for two intrahepatic cell types: the LSECs and the hepatocytes (38, 39).

Antigen presentation by isolated LSECs skews CD4⁺ T cell activation toward a regulatory phenotype with the expression of IL-4 and IL-10 (40). Priming of CD8⁺ T cells by LSECs resulted in incomplete activation with loss of IFN- and IL-2 cytokine production and a lack of cytotoxic activity. However, the exclusive role of LSECs in T cell priming has recently been questioned on the basis of experiments using LSECs that were isolated based on surface marker expression rather than counterflow elutriation. The problem is to determine whether low-level contamination of LSEC preparation by other APCs might have complicated the interpretation of previous results (41). Whether LSECs are finally agreed to be self-sufficient APCs does not impact directly on our present work, but would change the probability that they, rather than hepatocytes, are the important local intrahepatic APC.

Incomplete activation of CD8⁺ T cells with a lack of cytotoxicity and premature T cell death within the first few days after T cell priming has also been reported when transgenic antigen was expressed on hepatocytes (acting as nonprofessional APCs). However, continuous expression of a transgenic antigen is known to induce regulatory T cells, creating an alternative mechanism of tolerance. Similarly, the promiscuous gene expression of tissue-specific self-antigens in medullary thymic epithelial cells results in systemic central tolerance and formation of tolerogenic regulatory T cells (42–44). Therefore, modulation of CD8⁺ T cell responses by transgenic hepatic antigen could simulate mechanisms of self-tolerance and the control of autoimmunity rather than immune failure to an exogenous pathogen. In this work, antigen expression was introduced acutely by peptide injection, which would not be expected to induce thymic, self-specific regulatory T cell populations.

Our findings support a model in which a productive immune response is initially provoked by acute presentation of antigen within the liver, as seen in hepatic infections. In cases of antigen persistence, this immune response is modulated by regulatory systems over time, which might ultimately result in tolerance. This is reflected by the initial immune response in viral hepatitis infections preceding the chronic stage (45) or the immediate phase after liver transplantation with the occurrence of alloreactive lymphocytes before tolerance is established (7). The mechanisms through which such tolerance is established are still poorly understood. However, in this work we have tested and rigorously excluded the possibility that local, intrahepatic presentation of antigen to CD8⁺ T cells results in abortive interaction, maturation failure, and premature death. The reality is quite different. The interaction of CD8⁺ T cells with the liver results in the proliferation and maturation of CTLs with full effector function.

	MATERIALS AND METHODS

Top Abstract RESULTS DISCUSSION MATERIALS AND METHODS References

 
Mice.
Wild-type C57BL/6J and B6.SJL-Ptprc^a (B6.CD45.1) mice were purchased from The Jackson Laboratory. The bm8 mice were provided by L.R. Pease (Mayo Clinic, Rochester, MN). A colony of OT-I transgenic mice was maintained on a CD90.1 (Thy1.1) background. All mice were bred and housed in a specific pathogen-free environment and used between 6 to 10 wk of age. Experiments were performed in accordance with regulatory guidelines and standards set by the University Committee on Animal Resources of the University of Rochester.

Mouse liver transplantation.
Orthotopic mouse liver transplantation, initially reported by Qian et al. (46), was performed according to a technique described by Steger et al. (47) in the non-rearterialized version. The donor liver was obtained by dissection of the surrounding hepatic ligaments; the right adrenal vein, pyloric vein, and proper hepatic artery were ligated and divided. For continuous bile flow the gallbladder was ligated and removed. A polyethylene stent tube (inner diameter, 0.28 mm; SIMS Portex) was inserted into the lumen of the common bile duct and secured with 8–0 silk (Pearsalls). The infrahepatic inferior vena cava (IVC) and portal vein were clamped, and the organ was perfused with 10 ml of 4°C normal saline through the portal vein. The liver was removed and stored in 4°C PBS solution until transplantation. The transplantation procedure was performed under inhalation anesthesia with isoflurane. After clamping of the infrahepatic and suprahepatic IVC and the portal vein, the recipient's liver was completely removed and the donor organ was placed orthotopically into the abdominal cavity. The suprahepatic and the infrahepatic IVC were anastomosed with continuous running sutures using 10–0 nylon (Ethicon), and the portal vein was reconnected by cuff anastomosis. Reconstruction of the bile flow was achieved by inserting the graft's stent tube into the recipient's bile duct and securing it with three single 10–0 nylon sutures.

Radiation bone marrow chimera.
6–8-wk-old recipient mice were irradiated with a dose of 10 Gy (1,000 rad) using an RS2000 X-ray irradiator (RadSource Technologies). Depletion of mature T lymphocytes from donor bone marrow cells was achieved by incubation with anti-CD4 mAb (clone RL172.4) and anti-CD8 mAb (clone 3-16.8) and subsequent lysis using Guinea pig complement (Invitrogen). Recipient mice were injected with 10⁷ cells via the tail vein and were allowed to reconstitute for 28 d until additional experiments.

Cell isolation procedures.
For single cell suspensions, peripheral lymph nodes and spleens were mechanically homogenized between frosted glass slides. Liver leukocytes were isolated as described previously (48). In brief, livers were perfused with PBS, mashed through a cell strainer, and incubated for 40 min at 37°C in RPMI digestion buffer containing 0.05% collagenase IV and 0.002% DNase I (Sigma-Aldrich). The leukocyte population was obtained by density gradient centrifugation using a 22% Opti-prep (Axis-Shield) solution.

Flow cytometry and statistical analysis.
Cell solutions were stained for 20 min at 4°C with mAbs specific for TCR-ß, CD90.1, CD44, I-A^b, CD4, CD19, and IFN- (all from BD Biosciences); F4/80, CD8, and CD11b (all from Caltag Laboratories); and CD45.1, CD45.2, CD62L, and CD11c (all from eBioscience). For intracellular cytokine staining, the Cytofix/Cytoperm kit (BD Biosciences) was used according to the manufacturer's instructions after restimulation in the presence or absence of 1 µM SIINFEKL peptide for 6 h. Ungated cell samples of the described organs were acquired using a BD LSR II flow cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star) based on a lymphocyte size gate.

Adoptive transfer of OT-I cells and in vivo activation.
Transgenic CD8⁺ cells from spleens and lymph nodes were enriched by magnetic depletion of B cells, CD4⁺ T cells, dendritic cells, and NK cells using primary antibody (clone 212.Al specific for MHC class II molecules, clone GK1.5 specific for CD4, clone 2.4.G2 specific for FcRs, and clone HB.191 specific for NK1.1), followed by magnetic beads coated with secondary antibodies (QIAGEN). The purity of enriched CD8⁺ OT-I cells was >93% (±4%). To obtain a naive OT-I population, cells were sorted for a CD44^low CD62L^high phenotype using a FACSAria cell sorting system (BD Biosciences). The purity of CD8⁺ OT-I cells with a naive phenotype was 99% after sorting. 5 x 10⁶ OT-I cells were injected i.v. into transplant recipients and activated by daily i.p. injections of 25 nmol SIINFEKL peptide (New England Peptide) for 3 d starting 12 h after injection of OT-I cells.

In vitro proliferation assay.
Transplant recipients were injected i.p. for three consecutive days with 25 nmol SIINFEKL peptide. Cells from spleens and lymph nodes (cervical, axillary, brachial, inguinal, mesenteric, and celiac lymph nodes) were isolated by mechanical disruption. Bone marrow cells were obtained from femora and tibiae. Livers were perfused with a 37°C PBS solution containing 5% collagenase IV for 8–10 min, disrupted, and shaken to liberate intrahepatic cells into the media. To obtain an unbiased representation of all types of intrahepatic cells, the cell suspensions were simply washed, filtered to remove aggregates, and used in the assay without any further separation steps. To further differentiate APCs in the assay we fractionated such liver cells based on their CD45 expression by fluorescence- activated cell sorting using a FACSAria system (BD Biosciences). 3 x 10⁵ cells from spleens, lymph nodes, and bone marrow, as well as 2 x 10⁴ unseparated liver cells and 3 x 10⁵ fractionated liver cells were resuspended in culture media (DMEM/F12, 10% FBS, insulin-transferrin-selenium/ITS, and penicillin-streptomycin) and cocultured with 3 x 10⁵ CFSE-stained OT-I cells. Cultures were maintained for 96 h before flow cytometric analysis.

In vivo cytotoxicity assay.
Splenocytes from C57BL/6J mice were separated from the RBCs by density gradient centrifugation (Lympholyte-M; Cedarlane Laboratories), divided into two equal portions, and stained in a 2-or 0.2-µM CFSE solution (Invitrogen), respectively. Cell suspensions were pulsed for 1 h with either a 1-µM solution of SIINFEKL peptide as a specific target cell population (2 µM CFSE-stained cells) or a 1-µM solution of PA peptide as a control population (0.2 µM CFSE-stained cells). Specific target cells and control population were counted and mixed in a 1:1 ratio, and a total of 2 x 10⁷ cells was injected i.v. into transplant recipients. After 5 h, spleens, lymph nodes, and livers were harvested from recipient mice and analyzed by flow cytometry to determine the ratio of specific target cells versus control cells. Specific killing was calculated by the following formula: 100 – ([(percentage of SIINFEKL pulsed in immunized/percentage of PA pulsed in immunized)/(percentage of SIINFEKL pulsed in uninmmunized/percentage of PA pulsed in uninmmunized)] x 100).

Statistical analysis.
The statistical significance of the differences between groups of mice was tested using Student's t test. A value of P < 0.05 was considered significant.

Online supplemental material.
Fig. S1 depicts hepatic micro architecture and intrahepatic cell populations in transplanted and naive animals. Fig. S2 shows the fraction and subpopulations of radioresistant intrahepatic k^b+ wild-type leukocytes. Figs. S1 and S2 are available at http://www.jem.org/cgi/content/full/jem.20051775/DC1.