Liver Sinusoidal Endothelial Cells That Endocytose Allogeneic Cells Suppress T Cells with Indirect Allospecificity

Daisuke Tokita, Masayuki Shishida, Hideki Ohdan, Takashi Onoe, Hidetaka Hara, Yuka Tanaka, Kohei Ishiyama, Hiroshi Mitsuta, Kentaro Ide, Koji Arihiro and Toshimasa Asahara
J Immunol September 15, 2006, 177 (6) 3615-3624; DOI: https://doi.org/10.4049/jimmunol.177.6.3615

Abstract

A portal venous injection of allogeneic donor cells is known to prolong the survival of subsequently transplanted allografts. In this study, we investigated the role of liver sinusoidal endothelial cells (LSECs) in immunosuppressive effects induced by a portal injection of allogeneic cells on T cells with indirect allospecificity. To eliminate the direct CD4+ T cell response, C57BL/6 (B6) MHC class II-deficient C2tatm1Ccum (C2D) mice were used as donors. After portal injection of irradiated B6 C2D splenocytes into BALB/c mice, the host LSECs that endocytosed the irradiated allogeneic splenocytes showed enhanced expression of MHC class II molecules, CD80, and Fas ligand (FasL). Due to transmigration across the LSECs from BALB/c mice treated with a portal injection of B6 C2D splenocytes, the naive BALB/c CD4+ T cells lost their responsiveness to stimulus of BALB/c splenic APCs that endocytose donor-type B6 C2D alloantigens, while maintaining a normal response to stimulus of BALB/c splenic APCs that endocytose third-party C3H alloantigens. Similar results were not observed for naive BALB/c CD4+ T cells that transmigrated across the LSECs from BALB/c FasL-deficient mice treated with a portal injection of B6 C2D splenocytes. Adaptive transfer of BALB/c LSECs that had endocytosed B6 C2D splenocytes into BALB/c mice via the portal vein prolonged the survival of subsequently transplanted B6 C2D hearts; however, a similar effect was not observed for BALB/c FasL-deficient LSECs. These findings indicate that LSECs that had endocytosed allogeneic splenocytes have immunosuppressive effects on T cells with indirect allospecificity, at least partially via the Fas/FasL pathway.

It has been postulated that the liver might be a site for the induction of tolerance to exogenous MHC class II-restricted Ags that enter the organ in large numbers via portal circulation from the gut (1, 2). This phenomenon has been applied as a strategy to achieve the ultimate goal in transplantation; the induction of tolerance in T cells with allospecificity, i.e., single treatment with a portal venous injection of allogeneic cells succeeds in inducing persistent donor-specific tolerance across multiple minor histocompatibility and MHC class I incompatible barriers (3, 4). Such tolerance could be induced by injecting not only living allogeneic cells but also soluble Ags (5, 6), thereby increasing the possibility of efficiently inducing tolerance in T cells with indirect allospecificity. In addition to portal injection of donor cells, other treatments such as the administration of immunosuppressants (7, 8, 9, 10) and costimulatory blockades (11) are usually required to induce donor-specific tolerance across MHC class II incompatible barriers. This suggests that the tolerizing effects of portal injection of donor cells might be more efficient in T cells with indirect allospecificity than in T cells with direct allospecificity.

Portal injection-induced tolerance in T cells with indirect allospecificity might be a consequence of presentation of allopeptides by the host APCs in the liver to these T cells. The ability to present exogenous Ags on MHC class I or II molecules is restricted to Kupffer cells and dendritic cells in the liver. In portal injection-induced tolerance, the importance of Ag presentation by Kupffer cells is emphasized by the prevention of Ag sequestration and tolerance following the administration of gadolinium chloride (a rare earth metal that prevents Kupffer cell phagocytosis) (12, 13). Liver dendritic cells possess unique Ag-presenting properties and exhibit low expression of costimulatory molecules. Furthermore, the liver dendritic cells preferentially induce Th2 responses, suggesting that these cells mediate tolerogenicity (14, 15). In addition to Kupffer cells and liver dendritic cells, liver sinusoidal endothelial cells (LSECs),4 which line the hepatic sinusoids, are also capable of presenting soluble exogenous Ags to T cells that possess transgenic TCRs (16, 17, 18). Although a number of studies have demonstrated the importance of Ag presentation by Kupffer cells and liver dendritic cells in portal injection-induced transplantation tolerance, the role of Ag presentation by LSECs in such immune tolerance has not been investigated. In this study, we demonstrated that LSECs actively endocytose allogeneic splenocytes injected via the portal vein and that CD4+ T cells with indirect allospecificity lose their responsiveness on contact with such LSECs, at least partially via the Fas/Fas ligand (FasL) pathway. This study is the first to demonstrate that LSECs are capable of regulating a polyclonal population of nontransgenic T cells with certain specificity via the indirect pathway.

Materials and Methods

Mice

BALB/c (H-2d), C57BL/6 (B6) (H-2b), and C3H/HeN (C3H) (H-2k) mice (8- to 12-wk-old females) were purchased from Clea Japan. B6 MHC class II-deficient (C2D) (B6.129S2-C2tatm1Ccum) mice and BALB/c-FasLgld (BALB/c-gld) (Cpt.C3-Tnfsf6gld) mice were purchased from The Jackson Laboratory. All animals were maintained in a specific pathogen-free microisolator environment. Animal experiments were approved by the Institutional Review Board at Hiroshima University and were conducted in accordance with the guidelines of the National Institutes of Health (National Institutes of Health publication no. 86-23, revised 1985).

Portal venous injection of donor-type splenocytes

Splenocytes were prepared as a single-cell suspension after lyses of erythrocytes using an ammonium chloride/potassium solution. The splenocytes were irradiated (30 Gy) before treatment with a portal venous injection to eliminate possible graft-vs-host responses. Allogeneic splenocytes (30 × 106) in 0.5 ml of medium 199 (Sigma-Aldrich) containing 1% HEPES buffer were injected through the superior mesenteric vein using a 30-gauge needle.

Heterotopic heart transplantation

Donor-type heart allografts were transplanted. Cervical heterotopic heart transplantation was performed using the cuff technique modified from a previously described method (19). Briefly, the recipients were prepared before donor heart harvest to minimize the graft ischemic time. The right external jugular vein and the right common carotid artery were dissected free, mobilized as far as possible, and fixed to the appropriate cuffs. The cuffs were composed of polyethylene tubes (2.5F; Portex), the diameters of which were adjusted by physical extension. For anastomoses, the aorta and the main pulmonary artery of the harvested donor heart were drawn over the end of the common carotid artery and the external jugular vein, respectively. The graft ischemic time for the transplanted hearts was <30 min. The function of the grafts was monitored by daily inspection and palpation. Rejection was determined by the cessation of beating of the graft and was confirmed by histology.

Flow cytometry (FCM) analysis of anti-donor Abs

Indirect immunofluorescence staining of thymocytes from B6 mice was used to detect anti-donor-specific Abs. Cells (1 × 106) were incubated with 10 μl of serially diluted mouse serum, washed, and incubated with FITC-conjugated rat anti-mouse IgM and IgG mAb (BD Pharmingen), then were subjected to FCM analysis.

Isolation of LSECs

It has been reported that elevated levels of CD105 expression are selectively detected on the microvascular and vascular endothelial cells in regenerating tissue undergoing active angiogenesis (20, 21, 22). Similarly, LSECs that have an exceptional capacity for angiogenesis express CD105 in higher quantities than the endothelia of the central veins or other vessels in the liver (23). Therefore, CD105+ cells were positively selected for the isolation of LSECs from the nonparenchymal cell fraction of the liver as follows. Disaggregated liver cells were obtained from untreated BALB/c mice or BALB/c mice that had been treated with portal injection by the two-step collagenase perfusion method (24, 25) and were centrifuged at 50 × g for 1 min. The supernatant was centrifuged at 150 × g for 5 min. The pellet was resuspended, and the total cells obtained were stained with biotin-conjugated anti-CD105 (MJ7/18) (eBioscience). Subsequently, the cells were counterstained with streptavidin microbeads (Miltenyi Biotec) and magnetically sorted using an autoMACS cell sorter (Miltenyi Biotec). This sorting technique yielded 2–7 × 106 cells/body in the positive fraction. To analyze the purity of LSECs, aliquots of the sorted fractions were cultured in the presence of acetylated low-density lipoprotein (Ac-LDL)-BODIPY (final concentration, 15 mg/ml) (Molecular Probes) in a DMEM culture medium containing 10% heat-inactivated FBS, 5 mM 2-ME, 1% HEPES buffer, 100 IU/ml penicillin, and 100 mg/ml streptomycin on collagen I-coated 35-mm tissue culture dishes (BD Biosciences Labware). This fluorescence-labeled lipoprotein is exclusively taken up by endothelial cells such as LSECs. After 12 h, the cells were stained with anti-CD11b PE (M1/70) to detect the expression of CD11b as a marker for Kupffer cells. The sorted CD105+ cells were stained with anti-CD45 FITC (30F11.1) to detect the expression of CD45 as a marker for hemopoietic cells. These were purchased from BD Pharmingen.

Detection of LSECs that endocytose allogeneic splenocytes

B6 C2D splenocytes were washed in PBS and labeled with PKH26 (Sigma-Aldrich) for the detection of LSEC endocytosis. PKH26 labeling (2 × 10−6 M dye) was performed according to the manufacturer’s protocol. Following the portal injection of the irradiated B6 C2D splenocytes (30 × 106 cells) labeled with PKH26 into BALB/c mice, the host LSECs were isolated as described earlier. FcγR-blocking Abs (rat anti-mouse CD16/32 mAb) (2.4G2) were used, followed by staining with anti-mouse I-A/I-E FITC (2G9), anti-CD40 FITC (HM40-3), anti-CD80 FITC (16-10A1), anti-CD86 FITC (GL1) (BD Pharmingen), or unlabeled anti-mouse CD95 Ligand (MFL3) (eBioscience). The unlabeled mAb was visualized using anti-Armenian hamster IgG FITC (H+L) (eBioscience). The cells were analyzed by FCM analysis gating on the CD105+ population. The LSECs that endocytosed the injected splenocytes were identified as PKH26-labeled cells by FCM analysis. FCM was performed using a FACSCalibur (BD Biosciences). LSECs were also observed under a relief contrast confocal microscope.

Transendothelial migration assay

The isolated LSECs obtained from the experimental animals were applied onto a fibronectin-coated (50 μg/ml; Sigma-Aldrich) polycarbonate filter (pore size, 8 μm; Costar) containing a cell culture insert (1 × 105 cells/well in a 48-well plate). After 12 h of culture, nonadhered cells were removed from the filter membrane by washing. For the analysis of cell proliferation in indirect MLR, splenocytes were labeled with 5 μM CFSE (Molecular Probes) as described previously (26). CFSE-labeled syngeneic nonadherent splenocytes (10 × 106 cells/well) were overlaid on the monolayer of LSECs in the culture insert and incubated for 12 h. Splenocytes transmigrating to the bottom of the individual wells were harvested. These cells were used as responder cells for the subsequent indirect MLR (see Fig. 4⇓A).

Preparation of stimulator cells for indirect MLR

Donor-type B6 C2D or third-party C3H splenocytes (20 × 106 cells) were injected i.v. into BALB/c mice. Seven days after the injection, splenocytes were obtained from the BALB/c mice. These splenocytes were plated on 100-mm tissue culture dishes (Falcon 3003; BD Biosciences Labware). After incubation for 2 h, adherent cells were prepared as Ag-presenting stimulator cells for subsequent indirect MLR.

Indirect MLR assay

The Ag-presenting stimulator cells were irradiated with a dose of 30 Gy. CFSE-labeled responder cells were cultured with stimulator cells (in the ratio of 5:1) in a total volume of 2 ml of medium in a 24-well flat-bottom plate (BD Biosciences Labware) at 37°C in 5% CO2 in the dark for 5 days.

Quantification of T cell proliferation

In the indirect MLR using CFSE-labeled lymphocytes, proliferating T cells were detected by a multiparameter FCM analysis as described previously (27, 28). The harvested cells were stained with anti-mouse CD4 PE mAb (GK1.5) (BD Pharmingen). Alloreactivities of the responder T cells were quantified based on their CFSE-fluorescence intensities. In CFSE-fluorescence histograms, CD4+ T cells were selected by gating and were subsequently analyzed for CFSE fluorescence. Dead cells were excluded from the analysis by light scatter and/or by using propidium iodide. Theoretically, the CFSE-fluorescence intensity of cells that have undergone one cell division is half the value of the CFSE-fluorescence intensity of undivided cells. According to this theory, the number of divisions of alloreactive T cells could be mathematically determined by the logarithmic CFSE intensities on the basis of the peak at the extreme right (the peak of undivided cells). The limit of detection is seven or eight division cycles caused by the compression of peaks as the CFSE intensity approaches autofluorescent levels. Thus, divisions beyond six cycles are indistinguishable and are collectively referred to as division 7+. A single-cell dividing n times will generate 2n daughter cells. Using this mathematical relationship, the number of division precursors was extrapolated from the number of daughter cells of each division, and the number of times that mitotic events occurred in a CD4+ T cell subset was calculated. Using these values, mitotic indexes were calculated by dividing the total number of mitotic events by total precursors. Stimulation indexes were calculated by dividing the mitotic indexes of T cells responding to the splenic APCs pulsed with allogeneic cells by those of T cells responding to the control nonpulsed splenic APCs.

Intracellular cytokine staining for multiparameter FCM

Cytokine-secreting activity of T cells responding to allostimulation was determined as described previously (28). Following the indirect MLR culture, the cells were stimulated with 1 μM ionomycin (Sigma-Aldrich), 10 ng/ml PMA (Sigma-Aldrich), and GolgiPlug (a protein transport inhibitor containing brefeldin A) (BD Pharmingen) (2 μl in 2 ml of medium) for 4 h to enhance intracellular cytokine content without affecting cell proliferation, as described previously (29, 30, 31, 32). After harvesting, the cultured cells were stained with PerCP-CyChrome (PerCP-Cy5.5)-conjugated anti-CD4 (GK1.5) (BD Pharmingen). Nonspecific FcγR binding of labeled Abs was blocked by CD16/32 (2.4G2) (BD Pharmingen). Furthermore, the cells were stained with annexin V (BD Pharmingen) for gating out dead or apoptotic cells. Following cell surface Ag and annexin V staining, the cells were fixed and permeabilized using Cytofix/Cytoperm solution (containing 4% paraformaldehyde and saponin) (BD Pharmingen) and Perm/Wash buffer (containing FBS and saponin) (BD Pharmingen) according to the manufacturer’s instructions. For intracellular cytokine staining, PE-conjugated anti-IL-2 (JES6-1A12) and/or rat IgG1 (an isotype-matched control) were used.

Skin transplant model

Full-thickness tail skin was harvested from donor (B6 C2D) mice and grafted (2 pieces of ∼0.5 cm in size) onto the dorsal side of recipient (BALB/c) mice. Rejection was defined as the complete necrosis of the skin grafts. Skin grafts were completely rejected on a regular basis within 14 days after transplantation. Splenocytes from mice sensitized with skin allografts after rejection were labeled with CFSE. CFSE-labeled nonadherent splenocytes were used as sensitized responder cells for the subsequent indirect MLR.

Intraportal adaptive transfer of LSECs

LSECs from BALB/c mice that were either untreated or treated with a portal injection of irradiated B6 C2D splenocytes (30 × 106 cells/mouse) were isolated after 12 h of the injection as described earlier. These LSECs were prepared as a single-cell suspension. The LSECs (2 × 106 cells) in 0.5 ml of medium 199 containing 1% HEPES buffer were injected through the superior mesenteric vein by using a 30-gauge needle.

Statistical analysis

The results were statistically analyzed using the log-rank test or F test and Student’s t test of means, where appropriate. A p value of <0.05 was considered to be statistically significant.

Results

Portal venous injection of irradiated allogeneic splenocytes significantly prolonged the survival of subsequently grafted allogeneic hearts

To determine whether a portal venous injection of allogeneic cells could produce tolerance or hyporesponsiveness to allogeneic organ grafts, irradiated (30 Gy) splenocytes of either B6 MHC class II-deficient (C2D) or wild-type (WT) B6 mice were injected into BALB/c mice (30 × 106 cells/mouse) via the portal vein. Donor-type heart allografts were transplanted 7 days later. Survival curves of the grafted hearts are shown in Fig. 1⇓. All untreated BALB/c mice rejected WT B6 hearts within 7–9 days and C2D B6 hearts within 8–10 days (n = 5 and 4, respectively). However, a portal injection of WT B6 splenocytes induced indefinite WT B6 heart allograft survival in 60% of BALB/c mice (n = 5). A portal injection of C2D B6 splenocytes induced indefinite C2D B6 heart allograft survival in 80% of BALB/c mice (n = 5). Thus, the portal injection of the irradiated allogeneic splenocytes effectively led to an indefinite acceptance of subsequently transplanted donor-type heart allografts, particularly in the absence and even in the presence of donor MHC class II molecules. To determine whether the acceptance of heart allografts was due to tolerization of cells responding to alloantigens, the Ab levels against B6 alloantigens were determined in the sera of the BALB/c recipients. The serum levels of anti-B6 IgG Ab gradually elevated and reached a plateau several weeks after the cardiac allograft transplantation in the BALB/c mice that had received the portal injection of irradiated B6 splenocytes, even when both the splenocytes infused and the heart allograft lacked MHC class II molecules (data not shown). These findings indicate that the portal injection of irradiated allogeneic splenocytes in fact did not induce a long-lasting tolerance state in T cells and that the indefinite acceptance of the heart allograft resulting from the portal injection of irradiated allogeneic splenocytes might be derived from mechanisms other than persistent T cell tolerance.

FIGURE 1.
FIGURE 1.

A portal venous injection of the irradiated allogeneic splenocytes leads to the indefinite acceptance of subsequently transplanted donor-type heart allografts in the absence of direct allorecognition of donor MHC class II molecules. BALB/c mice were treated with a portal injection of either irradiated (30 Gy) allogeneic C2D or WT B6 splenocytes (30 × 106), and the donor-type heart allografts were subsequently transplanted 7 days later. A, Survival curves of WT heart allografts. Five untreated BALB/c mouse recipients were used as controls (dotted line), and five BALB/c mouse recipients were treated with a portal injection of irradiated B6 splenocytes (solid line). p < 0.01, untreated control BALB/c mice vs BALB/c mice treated with a portal injection of irradiated B6 splenocytes. B, Survival curves of C2D heart allografts. A portal injection of B6 C2D splenocytes prolonged the survival of subsequently transplanted heart allografts. Four untreated BALB/c mouse recipients were used as controls (dotted line), and five BALB/c mouse recipients were treated with a portal injection of irradiated B6 C2D splenocytes (solid line). p < 0.01, untreated control BALB/c mice vs BALB/c mice treated with a portal injection of irradiated B6 C2D splenocytes.

LSECs actively endocytosed allogeneic splenocytes injected via the portal vein

It could be possible that LSECs play a role in the immune regulatory effects induced by a portal injection of allogeneic cells on T cells with indirect allospecificity, regardless of the relevance of their role in the acceptance of subsequently transplanted heart allografts. It has been reported that LSECs show a large endocytic capacity for many ligands, including glycoproteins, components of the extracellular matrix, immune complexes, transferrin, and ceruloplasmin (33, 34, 35, 36, 37); however, further studies are required to elucidate whether LSECs endocytose allogeneic splenocytes after portal venous injection. For this purpose, we isolated CD105+ cells from BALB/c mice treated with a portal injection of irradiated B6 C2D splenocytes. The sorted CD105+ cells always contained >95% of CD11b− cells that had taken up Ac-LDL-BODIPY; these stained cells represented LSECs (Fig. 2⇓A). We also confirmed that the isolated CD105+ cells were completely free of contamination with CD45+ hemopoietic cells, which might have the capacity for Ag presentation. B6 C2D splenocytes were labeled with PKH26 before portal injection to identify the LSECs that endocytose the injected splenocytes as PKH26-labeled cells. Microscopic observation of the intracytoplasmic staining with PKH26 ruled out the possibility of the occurrence of doublets of LSECs along with adherents of the injected splenocytes or fragments (Fig. 2⇓B). At 12 h after the portal injection, ∼20% of LSECs had endocytosed the PKH26-labeled splenocytes (Fig. 2⇓C). In the phenotypic analyses, LSECs from untreated mice expressed low amounts of MHC class II, CD40, CD80, and CD86 phenotypes; these are surface molecules necessary for the efficient Ag presentation to T cells. LSECs that endocytosed the irradiated allogeneic splenocytes showed enhanced expression of MHC class II molecules and CD80, indicating their capacity for Ag presentation to CD4+ T cells (Fig. 3⇓). Notably, such LSECs concurrently lost CD40 expression and up-regulated FasL expression on their surface, suggesting the tolerogenic potential of the LSECs toward responding T cells.

FIGURE 2.
FIGURE 2.

LSECs actively endocytosed allogeneic splenocytes injected via the portal vein. A, Purity of isolated LSECs. The nonparenchymal cell fraction obtained from the liver of BALB/c mice that were either untreated or treated with a portal injection were stained with anti-CD105 mAb and sorted by an autoMACS cell sorter. To analyze the purity of LSECs, the sorted CD105+ cells were stained with anti-CD45 FITC (30F11.1) to detect the expression of CD45 as a marker for hemopoietic cells (left panel). Aliquots of the sorted fractions were cultured in the presence of Ac-LDL-BODIPY for 12 h, and the sorted cells were stained with anti-CD11b PE (M1/70) to detect the expression of CD11b as a marker for Kupffer cells (right panel). Representative FCM profiles are shown. The percentages indicate the total sorted CD105+ cells. B, The relief confocal microscopy image of CD105+ LSECs isolated from the host liver 12 h after the portal injection of irradiated B6 C2D splenocytes by positive selection using an autoMACS cell sorter (scale bar, 20 μm). B6 C2D splenocytes were labeled with PKH26 before portal injection into the BALB/c mice to identify the LSECs that had endocytosed the injected splenocytes as PKH26-labeled cells (red). C, Kinetics of the uptake of allogeneic-irradiated B6 C2D splenocytes by LSECs were analyzed by FCM. The percentages of PKH26-labeled phagocytic LSECs are shown for CD105+ cells isolated from the host liver.

FIGURE 3.
FIGURE 3.

Phenotypic characteristics of LSECs that endocytosed allogeneic splenocytes. Host LSECs were isolated 12 h after a portal injection of irradiated B6 C2D splenocytes (30 × 106) labeled with PKH26 into BALB/c mice. These were stained with anti-mouse I-A/I-E FITC, anti-CD40 FITC, anti-CD80 FITC, and FasL mAb. The LSECs that had endocytosed the injected splenocytes could be identified as PKH26-labeled cells by FCM analysis. The percentage of total sorted CD105+ cells in each fraction are shown. PKH26+ LSECs and PKH26− LSECs were selected by gating to compare the expression of various surface markers. The histogram reveals that PKH26+ LSECs (shaded histogram) expressed significantly higher levels of I-A/I-E, CD80, and FasL than the PKH26− LSECs (solid line). PKH26+ LSECs lost CD40 expression on their cell surface. The expression levels of each marker are presented as median fluorescence intensity. Average values ± SEM for the PKH26+ and PKH26− LSECs are shown. FCM analysis using anti-CD105 mAb revealed that the inocula of splenocytes prepared from B6 mice did not include CD105+ cells (<0.5%) (data not shown). The FCM profiles are representative of four independent experiments.

Induction of specific unresponsiveness in T cells with indirect allospecificity by transmigration across autologous LSECs that had endocytosed allogeneic splenocytes

The various functions of APCs (professional myeloid APCs) such as uptake, processing, and presentation of Ags are temporally and spatially separated. APCs endocytose peripheral Ags and then undergo maturation during their migration into the lymphatic tissue, where they encounter T cells in a specialized microenvironment. The LSEC has been described as a new type of organ-resident APC that executes all three salient functions of an APC simultaneously and exhibits immunomodulatory activity toward naive T cells (17, 18). It is possible that LSECs process the endocytosed allogeneic splenocytes and subsequently present alloantigens to the naive T cells.

We examined the effect of Ag presentation by LSECs to naive T cells with indirect allospecificity on the responsiveness of these T cells to a subsequent alloantigen presentation by professional APCs. For this purpose, we performed a transendothelial migration assay to mimic the structural features of the interaction between LSECs and T cells. In the liver, blood passes through a meshwork of sinusoids formed by LSECs. Therefore, circulating leukocytes frequently come in contact with LSECs owing to the small diameter (7–12 μm) of the sinusoids (38). Such a sinusoidal architecture is likely to promote the immunomodulatory activity of LSECs toward T cells. CFSE-labeled naive BALB/c nonadherent splenocytes first underwent transmigration across a monolayer (pores with a diameter of 8 μm) of LSECs from BALB/c mice that were either untreated or treated with a portal injection of irradiated allogeneic B6 C2D splenocytes; this enabled direct interaction between T cells and LSECs (Fig. 4⇓A). The transmigrated BALB/c lymphocytes were subsequently stimulated with splenic APCs from BALB/c mice that had been stimulated by treatment with an i.v. injection of the splenocytes from either donor-type B6 C2D or third-party C3H mice. The proliferative response of naive BALB/c CD4+ T cells to BALB/c APCs pulsed with B6 C2D splenocytes could be detected on FCM plots, although their stimulation indexes were lower than those usually observed in direct MLR assays in the fully allogeneic combinations. Such low stimulation indexes in indirect MLR assays are consistent with the previously reported fact that the frequency of T cells engaged in the indirect pathway of allorecognition is ∼100-fold lower than that of T cells participating in direct recognition (39). The enhanced proliferative response of the presensitized BALB/c CD4+ T cells (prepared by B6 C2D skin grafting) to BALB/c APCs pulsed with B6 C2D splenocytes proved the suitability of this indirect MLR assay. Nonetheless, the CD4+ T cells that had transmigrated across the LSECs from mice that were treated with a portal injection of irradiated B6 C2D splenocytes lacked the proliferative response to BALB/c APCs pulsed with B6 C2D splenocytes, while maintaining a normal response to BALB/c APCs pulsed with C3H splenocytes (Fig. 4⇓, B and C). Thus, CD4+ T cells that transmigrated across the Ag-presenting LSECs that had endocytosed allogeneic splenocytes were rendered unresponsive to alloantigens via indirect recognition in an Ag-specific manner. The proliferative response of the BALB/c CD8+ T cells that transmigrated across the BALB/c LSECs to BALB/c APCs pulsed with B6 C2D splenocytes was undetectable even on FCM plots, regardless of whether pretreatment with a portal injection of B6 C2D splenocytes was conducted (data not shown). This might be attributed to the much lower number of CD8+ T cells with indirect allospecificity than those of CD4+ T cells with indirect allospecificity, as demonstrated previously (40).

FIGURE 4.
FIGURE 4.

CD4+ T cells with indirect allospecificity were rendered unresponsive to alloantigens on contact with autologous LSECs that had endocytosed the respective alloantigens. A, System of transendothelial migration assay. Seven days after the portal injection, the LSECs were isolated from BALB/c mice that were either untreated or treated with a portal injection of irradiated allogeneic B6 C2D splenocytes. The isolated LSECs were applied onto the cell culture insert of a polycarbonate filter that was fibronectin-coated and had a pore size of 8 μm. After 12 h of culture, the nonadhered cells were removed from the polycarbonate filter membrane by washing. CFSE-labeled naive BALB/c nonadherent splenocytes first underwent transmigration across a monolayer of LSECs from BALB/c mice that were either untreated or treated with a portal injection of irradiated allogeneic B6 C2D splenocytes; this enabled direct interaction between T cells and LSECs. The transmigrated BALB/c lymphocytes were subsequently stimulated with splenic APCs from BALB/c mice that had been treated with an i.v. injection of splenocytes from either donor-type B6 C2D or third-party C3H mice (indirect MLR). CFSE-labeled responder cells (2 × 106) were cultured with 4 × 105 irradiated stimulator cells in the ratio of 5:1 for 5 days. After the indirect MLR, the harvested lymphocytes were stained with PE-conjugated anti-mouse CD4 mAb. Subsequently, T cell proliferation (division) was visualized by FCM analysis as the serial halving of CFSE-fluorescence intensity. B, Representative FCM results of CFSE-labeled CD4+ T cell division in the subsequent indirect MLR. C, Stimulation indexes of CD4+ T cells with indirect allospecificity in the subsequent indirect MLR are shown. Each point represents an individual mouse, and the average values of four independent mice in each group are shown. ∗, p < 0.05; ∗∗, p < 0.01.

Exposure to LSECs that endocytose alloantigens attenuated the IL-2-secreting activity of CD4+ T cells with indirect allospecificity

We have previously reported a method that combines MLR, which uses a CFSE-labeling technique, intracellular cytokine immunofluorescence staining, and multiparameter FCM analysis for the simultaneous determination of proliferation and cytokine-secreting activity of T cells responding to allostimulation (28). Using this technique, IL-2-secreting cells were detected in proliferating BALB/c CD4+ T cells in response to BALB/c APCs pulsed with B6 C2D splenocytes (Fig. 5⇓). Transmigration across the LSECs from mice that were treated with a portal injection of irradiated B6 C2D splenocytes significantly reduced the frequency of IL-2-secreting cells in proliferating BALB/c CD4+ T cells in response to BALB/c APCs pulsed with B6 C2D splenocytes. In contrast, the frequency of the IL-2-secreting cells remained high in proliferating BALB/c CD4+ T cells in response to BALB/c APCs pulsed with C3H splenocytes. Thus, the exposure to LSECs that endocytose donor-type alloantigens attenuated not only the proliferating activity but also the IL-2-secreting activity of CD4+ T cells with indirect allospecificity.

FIGURE 5.
FIGURE 5.

Exposure to LSECs that endocytose alloantigens attenuated the IL-2-secreting activity of CD4+ T cells with indirect allospecificity. The transmigrated BALB/c CFSE-labeled responder lymphocytes were cultured with irradiated stimulator splenic APCs from BALB/c mice that had been stimulated with an i.v. injection of splenocytes from either donor-type B6 C2D or third-party C3H mice. The indirect MLR-cultured cells were stained with PerCP-Cy5.5-conjugated anti-CD4, followed by staining with allophycocyanin-conjugated annexin V. Subsequently, the cells were fixed, permeabilized, and stained with PE-conjugated IL-2 mAbs or isotype-matched control Abs (IgG1). A four-colored FCM was performed to determine the proliferation and cytokine-secreting activity in the MLR. CD4+ T cells were selected by gating and analyzed for IL-2-secreting activity. A, Representative FCM results of CFSE-labeled CD4+ T cell division in the indirect MLR. The FCM profiles shown are representative of four independent experiments. The number refers to the percentage of total cells in each quadrant. B, The frequency of IL-2-producing cells in the proliferated CD4+ T cell fractions. The frequency (%) was calculated using the quadrant data of the FCM contour plots in A according to the following formula: percentage of the upper left/(percentage of the upper left + percentage of the lower left) × 100. Average values ± SEM for four independent experiments are shown. ∗∗, p < 0.01.

CD4+ T cells that transmigrated across the LSECs that had endocytosed allogeneic splenocytes were rendered unresponsive to alloantigens by a mechanism involving Fas/FasL interaction

Based on the results of phenotypic studies showing enhanced expression of FasL on the LSECs that endocytose allogeneic splenocytes and the previously reported result that FasL engagement inhibits CD4+ T cell proliferation, cell cycle progression, and IL-2 secretion (41, 42, 43), we assumed that such LSECs inhibited allospecific T cell proliferation via apoptosis through Fas/FasL interaction. Consistent with this hypothesis, the transmigration of naive BALB/c CD4+ T cells across the LSECs from FasL-deficient BALB/c-gld mice treated with a portal injection of B6 C2D splenocytes failed to induce unresponsiveness in allospecific T cells (Fig. 6⇓). This observation ruled out the possibility that the loss of ability of BALB/c CD4+ T cells to respond to BALB/c APCs pulsed with B6 C2D splenocytes was merely due to the adhesion of the responding BALB/c CD4+ T cells to LSECs.

FIGURE 6.
FIGURE 6.

CD4+ T cells that transmigrated across the LSECs that had endocytosed allogeneic splenocytes lost their responsiveness to alloantigens by a mechanism involving Fas/FasL interaction. CFSE-labeled BALB/c splenocytes first underwent transmigration across the LSECs from BALB/c WT (•; n = 3) or BALB/c-gld FasL-deficient mice (▪; n = 3) that were treated with a portal injection of irradiated B6 C2D splenocytes. The transmigrating cells were subsequently stimulated with the irradiated splenic APCs from BALB/c mice that had been pulsed with an i.v. injection of donor-type B6 C2D splenocytes (indirect MLR). The inhibition rate of indirect alloimmune response by the LSECs was represented by the percentage of stimulation index of CD4+ T cells transmigrating across the LSECs from the untreated control BALB/c mice. ∗, p < 0.05.

Adaptive transfer of BALB/c LSECs that had endocytosed B6 C2D splenocytes into BALB/c mice via the portal vein prolonged the survival of subsequently transplanted B6 C2D hearts

Next, we investigated the biological significance of the mechanism of LSECs that was described earlier, and the relevance of such a mechanism with regard to the acceptance of heart allografts after a portal injection of allogeneic cells. LSECs obtained from BALB/c mice 12 h after the portal injection of irradiated B6 C2D splenocytes were adaptively transferred via the portal vein into BALB/c mice (2 × 106 cells/mouse). After 7 days, allograft hearts from B6 C2D mice were transplanted into BALB/c mice. Adaptive transfer of LSECs from BALB/c mice treated with a portal injection of irradiated B6 C2D splenocytes prolonged the survival of subsequently transplanted allograft hearts, although this effect was not long lasting. The prolonging effect of adaptive transfer of LSECs from BALB/c-gld mice treated with a portal injection of irradiated B6 C2D splenocytes on the survival of subsequently transplanted allograft hearts was significantly less than that of LSECs from WT BALB/c mice treated similarly (Fig. 7⇓). Thus, the immunosuppressive effect of LSECs on T cells with indirect allospecificity via the Fas/FasL pathway is involved in the mechanism underlying the prolongation of heart allograft survival after a portal injection of allogeneic cells, at least in the early phase.

FIGURE 7.
FIGURE 7.

Adaptive transfer of BALB/c LSECs that had endocytosed B6 C2D splenocytes into BALB/c mice via the portal vein prolonged the survival of subsequently transplanted B6 C2D hearts. LSECs were obtained from either untreated BALB/c mice, BALB/c mice 12 h after a portal injection of irradiated (30 Gy) B6 C2D splenocytes (30 × 106 cells/mouse), or BALB/c-gld FasL-deficient mice 12 h after a portal injection of irradiated B6 C2D splenocytes. The isolated LSECs were adaptively transferred via the portal vein into BALB/c mice (2 × 106 cells/mouse), and the allograft hearts from B6 C2D mice were transplanted 7 days later. Survival curves of B6 C2D heart allografts are shown. Five BALB/c mice received untreated BALB/c LSECs (dotted line), five BALB/c mice received BALB/c LSECs that had endocytosed B6 C2D splenocytes (solid line), and three BALB/c mice received BALB/c-gld LSECs that had endocytosed B6 C2D splenocytes (bold line). p < 0.01, recipients of untreated BALB/c LSECs vs those of BALB/c LSECs that had endocytosed B6 C2D splenocytes. p < 0.02, recipients of BALB/c LSECs that had endocytosed B6 C2D splenocytes vs those of BALB/c-gld LSECs that had endocytosed B6 C2D splenocytes.

Discussion

The liver appears to favor the induction of immune tolerance rather than immunity. A number of observations demonstrate that Ag-specific immune tolerance is the result of Ag presentation in the liver. Allogeneic liver transplants are often well accepted by a recipient (44, 45, 46), leading to tolerance to further organ transplants from the same donor but not to third-party grafts (47, 48). We have recently demonstrated a novel relevant mechanism of such liver allograft tolerance, i.e., naive allogeneic LSECs selectively tolerize CD4+ and CD8+ T cells with direct allospecificity in mice in which liver allografts are normally accepted without recipient immune suppression across MHC barriers (49). In allogeneic mixed hepatic constituent cell-lymphocyte reaction (MHLR) assay, whole constituent cells did not promote T cell proliferation. When LSECs were depleted from the hepatic constituent cell stimulators in the allogeneic MHLR assay, a marked proliferation of reactive CD4+ and CD8+ T cells was observed. After restimulation with irradiated BALB/c splenocytes, we observed nonresponsiveness of B6 T cells that had transmigrated across allogeneic BALB/c LSECs and marked proliferation of T cells that had transmigrated across syngeneic B6 or third-party SJL/j LSECs. These results raised the question of whether a similar mechanism involving LSECs can explain another well-known phenomenon of increased graft acceptance after a pretransplant portal venous injection of donor leukocytes (3, 4).

The LSEC has been described as a new type of APC that induces immune tolerance in naive T cells (17, 18). Furthermore, LSECs are capable of stimulating naive CD4+ T cells. However, following priming by Ag-presenting LSECs, CD4+ T cells fail to subsequently differentiate into Th1 phenotype, instead they differentiate into regulatory T cells that express IL-4 and IL-10 on restimulation (6, 50). LSECs also have the capacity to present exogenous Ags on MHC class I molecules to CD8+ T cells, a process termed as cross-presentation (5). Initially, the stimulation of naive CD8+ T cells by LSECs results in the proliferation of T cells and the release of cytokines. However, finally, it leads to Ag-specific tolerance, as demonstrated by the simultaneous loss of cytokine expression and the failure of CD8+ T cells to develop into cytotoxic effector T cells. However, such immune regulatory effects of LSECs have been observed only in a model in which the interaction of soluble exogenous Ags and its corresponding transgenic TCRs occurs. Thus, the capacity of LSECs to regulate a polyclonal population of nontransgenic T cells with allogeneic specificity remains to be elucidated.

We demonstrated that host LSECs actively endocytose allogeneic splenocytes injected via the portal vein. Host LSECs that endocytose allogeneic splenocytes highly expressed MHC class II molecules and CD80 costimulatory molecules, probably due to the processing of endocytosed allogeneic cells and presentation of alloantigens. It is possible that the LSECs process the endocytosed allogeneic splenocytes and subsequently present alloantigens to naive CD4+ T cells through interaction between autologous MHC class II molecules and TCRs. Such Ag presentation by LSECs might negatively regulate CD4+ T cells with indirect allospecificity. The cumulative surface area of LSECs is very large, and hepatic microcirculatory parameters allow frequent contact between LSECs and passenger leukocytes. Considering the large volume of blood that passes through the liver daily, it is probable that LSECs are ideally positioned within the liver to establish peripheral immune tolerance (17, 51). We conducted a T cell transendothelial migration assay to mimic the structural features of the interaction between LSECs and T cells. In this system, the responsiveness of naive CD4+ T cells to stimulus with syngeneic splenic APCs pulsed with allogeneic C2D splenocytes was abrogated by transmigration across LSECs that endocytosed allogeneic C2D splenocytes; this indicates that T cells with indirect allospecificity could be negatively regulated by direct contact with LSECs that present alloantigens (Fig. 4⇑). We also demonstrated that the up-regulation of FasL expression on LSECs that endocytosed allogeneic cells could contribute to their immunosuppressive potential on alloantigen recognition via the indirect pathway. The deficiency of FasL on LSECs in mutant mice significantly attenuated their suppressive property toward CD4+ T cells with indirect allospecificity (Fig. 6⇑). Because there appears to be a residual inhibitory effect of LSECs on T cells even in the absence of FasL, mechanisms other than the Fas/FasL pathway may also be involved in the LSEC-induced immunosuppression of CD4+ T cells. Based on the previously reported results demonstrating the importance of CD40/CD154-mediated costimulation in indirect presentation models (52, 53), insufficient expression of costimulatory molecules, i.e., significant down-regulation of CD40 expression on the LSECs that endocytosed allogeneic cells (Fig. 3⇑), might contribute to their immunosuppressive effects.

Endothelial cells have been shown to activate T cell responses toward alloantigens, thereby triggering transplant rejections (54). However, recently, it has been reported that endothelial cells exposed to IL-10, IFN-α, and/or vitamin D3 induce the expression of Ig-like transcript (ILT)3 in endothelial cells, thereby tolerizing them (55). ILT3 belongs to a family of Ig-like inhibitory receptors that are structurally and functionally related to killer cell inhibitors and contains ITIMs (56, 57, 58) that mediate the inhibition of cell activation by recruiting tyrosine phosphatase Src homology protein-1 (57). Because various hemopoietic cells (i.e., intrahepatic macrophages, dendritic cells, NK cells, NK T cells) that reside in the liver have a capacity to release anti-inflammatory mediators, including IL-10, the expression of ILT3 in LSECs might be induced by factors unique to the liver microenvironment. The possibility of such an alternative explanation for LSEC-induced immunosuppression of T cells with indirect allospecificity remains to be elucidated.

In this study, although we proved that T cells with indirect allospecificity lose their reactivity on exposure to LSECs that endocytose alloantigens, at least partially through Fas/FasL interaction, the relevance of such a mechanism with regard to the acceptance of heart allografts after a portal injection of allogeneic cells remains unclear. The adaptive transfer of BALB/c LSECs that had endocytosed B6 C2D splenocytes into BALB/c mice prolonged the survival of subsequently transplanted B6 C2D hearts; however, this effect was not long lasting. This indicates that the immunosuppressive effect of LSECs on T cells plays a significant role at least in the early phase after the portal injection of allogeneic cells. However, further investigations are required to clarify the responsibility of LSECs for persistent acceptance of heart allografts after a portal injection of allogeneic cells. In the present study, MHC class II-deficient heart allografts appeared to be more susceptible to persistent acceptance induced by a portal injection of allogeneic cells than MHC class II-expressing heart allografts. It is possible that the immunosuppressive effect of the portal injection of donor cells is more efficient in T cells with indirect allospecificity than in T cells with direct allospecificity. Alternatively, it is also possible that grafts that do not express MHC class II molecules merely survive longer than grafts that express these molecules. In either case, the influence of a portal injection of donor cells on T cells with direct allospecificity remains to be elucidated. Previous studies have shown a prolonged allograft survival after an intrathymic injection of donor MHC peptides. This suggests that the manipulation of the indirect pathway can alter the course of rejection when both direct and indirect responses are available (59, 60); however, the precise mechanism remains unknown. Thus, it is possible that the unknown mechanisms underlying hyporesponsiveness toward T cells with indirect allospecificity induced by the portal injection of allogeneic cells simultaneously inhibits T cell responses via the direct alloantigen presentation pathway. For example, if the release of immunosuppressive cytokines mediated by the LSECs is also involved in the mechanisms of their tolerance toward T cells with indirect allospecificity, this may prevent T cell responses via the direct alloantigen presentation pathway. Further studies are required to clarify this concept.

Acknowledgments

We thank Drs. H. Tashiro, M. Ochi, and S. Kishida for their advice and encouragement.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This study was supported in part by a Grant-in-Aid for Exploratory Research (no. 17659389) of the Ministry of Education, Culture, Science and Technology from the Japan Society for the Promotion Science.

  • ↵2 D.T. and M.S. contributed equally to this work and should be considered as first authors.

  • ↵3 Address correspondence and reprint requests to Dr. Hideki Ohdan, Department of Surgery, Division of Frontier Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-Ku, Hiroshima 734-8551, Japan. E-mail address: hohdan{at}hiroshima-u.ac.jp

  • ↵4 Abbreviations used in this paper: LSEC, liver sinusoidal endothelial cell; FasL, Fas ligand; Ac-LDL, acetylated low-density lipoprotein; FCM, flow cytometry; WT, wild type; MHLR, mixed hepatic constituent cell-lymphocyte reaction; ILT, Ig-like transcript.

  • Received December 23, 2004.
  • Accepted June 28, 2006.

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