Systemic transmigration of allosensitizing donor dendritic cells to host secondary lymphoid organs after rat liver transplantation

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Donor dendritic cell (DC) migration and allosensitization in host secondary lymphoid organs after liver transplantation are ill defined. We used rat models to investigate graft-derived cells and intrahost allosensitization. Liver transplantation induced diffuse blood-borne migration of donor major histocompatibility class II antigen–positive (MHCII+) cells and MHCI+ cells from the graft to host secondary lymphoid organs, not only the spleen, but also lymph nodes and Peyer's patches. The migrated MHCII+ cells included DCs and some T cells and B cells. The DCs formed clusters with host BrdU+ cells where they up-regulated CD86+, and a CD8+ T cell proliferative response originated within 24 hours after liver transplantation, demonstrating that these DCs can quickly mature and trigger direct allosensitization in host lymphoid organs. Transfer of allogeneic bone marrow cells also induced DC transmigration and a similar host response. In contrast, allogeneic thoracic duct lymph cells contained many fewer transmigrating DCs, and their transfer induced a comparable T cell response but significantly weaker CD8+ T cell proliferation. Thus, there is a different outcome via the indirect pathway by host DCs that have captured donor alloantigens. Conclusion: The rat liver as well as bone marrow contains an immature DC population that can systemically transmigrate through blood vessel walls of the host secondary lymphoid organs, quickly mature, and induce diffuse intrahost CD8+ T cell responses, which may promote graft rejection. (HEPATOLOGY 2008.)

The liver is one of the most leukocyte-rich organs and contains lymphoid cells, dendritic cells (DCs),1, 2 and myeloid lineages including immature hematopoietic cells.3 When liver transplantation is performed, these cells may enter the host blood circulation and induce alloresponse in host lymphoid organs as passenger leukocytes. Donor DCs in the graft express a high level of self major histocompatibility complex (MHC) antigens which are recognized as alloantigens by host T cells (direct allosensitization or pathway).4, 5 Host DCs may take up graft MHC antigens and present them to host T cells (indirect pathway or allosensitization).4, 5 Accordingly, if donor DCs enter the blood, they may induce intrahost direct allosensitization.

Previously, we found that allogeneic rat heart transplantation induced the blood-borne migration of donor DCs to the host spleen and hepatic lymph nodes (LNs) via the liver.6 These migrated DCs formed clusters with DNA-synthesizing (5-bromo-2′-deoxyuridine [BrdU]-positive [BrdU+]) host T cells (BrdU+ cluster) in which the T cell proliferative response begun, representing the sites of direct allosensitization.4 Furthermore, host interdigitating DCs captured donor MHC antigens and formed BrdU+ clusters with host proliferating cells, suggesting an indirect pathway.4 Therefore, the BrdU+ cluster formation by donor or host DCs in situ could be a hallmark indicating that the alloresponse is ongoing there.

By using a rat liver allotransplantation model, Demetris et al.7 reported the migration of donor class II MHC-positive (MHCII+) cells to the host spleen and LNs. This group also detected cluster formation between donor MHCII+ cells and donor MHCII proliferating cells in the spleen, suggesting that the migrated cells induced the intrahost allosensitization. However, these migrated cells were not defined as DCs. More recently, in a similar rat model, a few migrated donor MHCII+ cells in the spleen were further identified as CD11c+ DCs8 but neither BrdU+ cluster formation nor other lymphoid organs were examined. Thus, donor DC migration and the allosensitization pathway in the host secondary lymphoid organs after liver transplantation are still ill-defined.

The aim of the present study is to investigate the phenotype and kinetics of graft liver-derived cells and the manner of the intrahost allosensitization by using a fully allogeneic rat liver transplantation model. Bone marrow cells (BMCs) of the same allogeneic combination were intravenously transferred and used as a positive control for the passenger leukocytes that contain a DC population.9 As a negative control of the passenger leukocytes, thoracic duct lymph cells (TDLs) containing mostly the recirculating lymphocytes and a very few DCs, approximately 0.2% of rat TDL,10 were intravenously transferred. Although the positive control may induce direct allosensitization by the donor DCs, the negative control should induce only the indirect pathway by the host DCs that have captured the donor MHC antigens. Because alloreactive CD8+ T cells are mainly responsible for acute graft rejection,11 we compared CD8+ T cell responses among these 3 groups.

We report here that the liver as well as bone marrow, but not TDLs, contain immature DCs that can systemically transmigrate to the host lymphoid organs, quickly mature, and induce diffuse intrahost CD8+ T cell alloresponses.

Abbreviations

BMC, bone marrow cell; BrdU, 5-bromo-2′-deoxyuridine; DC, dendritic cell; FACS, fluorescence-activated cell sorter; GvHR, graft-versus-host reaction; HvGR, host-versus-graft reaction; LN, lymph node; mAb, monoclonal antibody; MHCI, class I major histocompatibility complex; MHCII, class II major histocompatibility complex; TCR, T cell receptor; TDL, thoracic duct lymph cell.

Materials and Methods

Animals.

Inbred ACI rats (RT1AaBa) were supplied by National Bio Resource Project for the Rat in Japan, Kyoto University (Kyoto, Japan). Lewis rats (RT1AlBl) were supplied by Charles River Japan Inc. (Tsukuba, Japan). They were reared under specific pathogen-free conditions. Handling and care were in accordance with the University of Dokkyo's Regulation for Animal Experiments and Japanese Governmental Law (No. 105) and approved by the Dokkyo Medical University Animal Experiment Committee. ACI rats donated liver grafts, BMCs, and TDLs; Lewis rats were recipients.

Liver Transplantation.

Orthotopic liver transplantation from ACI to Lewis rats was performed using a cuff method under ether anesthesia. A simplified cuff was used for the portal and intrahepatic caval anastomoses.12 A Teflon tube stent was used for the biliary anastomosis. The hepatic artery was not reconstructed because grafts were functionally competent without an arterial supply.12

Lewis rats rejected ACI hepatic allografts with a mean graft survival time of 10.1 ± 0.7 days,12 whereas control Lewis-to-Lewis isografts survived indefinitely. At various times from 12 hours to 7 days after transplantation, host rats received an intravenous injection of BrdU (2 mg/100 g body weight; Sigma Chemical Co., St. Louis, MO) and were sacrificed 1 hour later. Graft livers and host secondary lymphoid organs including the spleen, cervical LNs, parathymic LNs, mesenteric LNs, celiac LNs, and Peyer's patches were excised and fresh-frozen for immunostaining. Isograft control rats were sacrificed on day 2 after transplantation.

Adoptive Cell Transfer.

BMCs were collected aseptically from the femura and humeri. TDLs were obtained by routine thoracic duct cannulation13 and aseptically collected overnight at 4°C. Cell viabilities were greater than 90% for BMCs and greater than 95% for TDLs, as determined by the trypan blue dye exclusion test. Lewis rats received intravenous injection of 2.5 × 108 whole BMCs (BMC transfer group) or 1 × 108TDLs (TDL transfer group) from ACI rats. Host rats were sacrificed on days 1-7 after cell transfer, and tissues were fresh-frozen as above. Because many fewer cells migrated to host lymphoid organs in the BMC transfer group versus the liver transplant and TDL transfer groups, cell dose was selected to yield migration comparable to the liver transplant group.

Antibodies and Reagents.

Monoclonal antibodies (mAbs) and labeled secondary antibodies used for immunohistology and fluorescence-activated cell sorter (FACS) analysis are listed in Table 1. Some mAbs were purified from culture supernatants and coupled to fluorescein isothiocyanate, R-phyocoerythrin (Dojin, Kumamoto, Japan), Alexa-Fluor 350, 488, 594, or 647 (Molecular Probes, Eugene, OR), or biotin (Pierce, Rockford, IL).

Table 1. Antibodies Used in This Study
1st Ab
AntigenCloneSourceConjugate
  1. a BD Pharmingen, b CALTAG, c CEDARLANE, d ECACC, e LSL, f Oxford Biotech, g Serotec, h R-phycoerythrin, i fluorescein isothiocyanate, j alkaline phosphatase, k horseradish peroxidase, l 7-amino-4-methylcoumarin-3-acetic acid, * self conjugation

CD31F4aR-PEh
CD8β341g 
CD11c8A2gR-PE*
CD45R (B220)HIS24gFITC,i R-PE
CD49d (α4 integrin)MRα4-1aFITC
CD62LOX85cR-PE
CD8624FbR-PE
CD103 (αE integrin)OX62gunconjugated,R-PE*
CD163ED2gunconjugated,R-PE*
Ig κ chainOX12g 
Ig λ chainMARL4g 
Ig μ chainMARM4gFITC,R-PE*
RT1.Aa (donor MHCI)MN4-91-6dBiotin*,R-PE*,Alexa 594*
RT1.Ba/c (donor MHCII)OX76dAlexa 594*,647*
RT1.Bl (host MHCII)OX3dAlexa 488*,594*
TCRαβR73dFITC,R-PE*
BrdUBU1/75f 
Type IV collagen(polyclonal)e 
2nd Ab
ProductSource Conjugate
goat Ig to mouse IgSigma,A9316 ALP j
sheep F(ab′)2 to rat IgSigma,A4812 ALP
sheep F(ab′)2 to mouse IgSigma,F2266 FITC
rabbit Ig to mouse IgDako,P161 HRP k
goat F(ab′)2 to rabbit IgCappel,55693 HRP
donkey F(ab′)2 to mouse IgJackson,715-116-151 R-PE
donkey Ig to rabbit IgJackson,711-155-152 AMCA l

FACS Analysis.

Peyer's patches and spleens of host rats and untreated donor livers were digested with collagenase D (Roche Diagnostics, Indianapolis, IN), and cells were isolated as described.14 Liver nonparenchymal cells and splenocytes were purified by a density gradient separation using OptiPrep (AXIS-SHIELD, Oslo, Norway). The number of isolated nonparenchymal cells was 4.6 × 107 ± 1.1 × 107 cells/donor liver (n = 4). For analysis of donor cell migration, single-cell suspensions of spleen and Peyer's patches were preincubated with FcγIIR-blocking mAb (clone D34-485; BD Biosciences, San Jose, CA). After incubation with biotin-conjugated anti-donor MHCI mAb and streptavidin magnetic microbeads (Miltenyi Biotec, Belgisch Gladbach, Germany), donor cells were positively isolated by autoMACS (Miltenyi Biotec). The isolated cells were stained for anti-donor MHCII-Alexa Fluor 647–conjugated and R-phyocoerythrin–conjugated mAbs. Cells were washed twice and analyzed on a FACS Calibur (BD Biosciences) using Cell Quest software (BD Biosciences). Dead cells were excluded by using propidium iodide. To detect resident DCs in the donor tissue, liver nonparenchymal cell suspension was analyzed on a FACS as above.

Immunohistological Analysis.

Double or triple immunostaining of fresh 4-μm-thick cryosections was performed as described.4, 6 The T cell areas of the spleen, LN, and Peyer's patches correspond to the inner portion of the periarterial lymphocyte sheath, the paracortex, and the interfollicular area, respectively. The B cell areas of all these organs are the lymph follicles. Type IV collagen was often immunostained to reveal tissue framework.4

To examine the in situ proliferative response in the host secondary lymphoid organs, the number of BrdU+ cells/mm2 of T cell area was counted. During the intragraft host cell response, infiltrated host cells accumulated mainly in the portal area and hepatic vein area. Accordingly, both areas were defined as the nonsinusoidal area (outside the sinusoidal area of the liver lobules1), and the number of BrdU+ cells/mm2 of the nonsinusoidal area was counted.

To analyze cluster formation by donor or host MHCII+ cells and proliferating cells, donor MHCII+ cells clustering with either one BrdU+ cell or two or more BrdU+ cells in the splenic T cell area were counted. To investigate donor cell proliferation and clustering with host MHCII+ cells, we determined the number of BrdU+ donor MHCI+ cells and the proportion of BrdU+ donor MHCI+ cells clustering with host MHCII+ cells relative to total BrdU+MHCI+ cells in the splenic T cell area. For phenotype analysis of donor MHCII+ cluster-forming cells, serial fresh 2 μm-thick cryosections of Peyer's patches harvested on day 2 were prepared. One section was stained for donor MHCII, type IV collagen, and BrdU. A neighboring section was stained either for CD11c, CD86, or host MHCI; type IV collagen; and BrdU. The corresponding areas of two neighboring sections were photomicrographed, and the proportion of either CD11c+, CD86+, or host MHCI donor MHCII+ cluster-forming cells was calculated. More than 15 clusters per rat and 3 rats for each phenotype were examined.

To analyze the CD8+ T cell response, spleen cryosections harvested on days 1-4 after liver transplantation or cell transfer were examined. A mAb to CD8β was used, because rat natural killer cells are also CD8α+. Cells with a red BrdU+ nucleus thoroughly outlined by a blue CD8 β + cell membrane were registered as CD8+ proliferating cells. BrdU+ cells with a discontinuous CD8+ cell membrane were determined as CD8. The number of BrdU+CD8+ cells and total BrdU+ cells/mm2 in the T cell area were counted.

Statistical Analysis.

Each parameter was measured in a blinded fashion and expressed as the mean ± standard deviation (n = 3-4 rats). For estimation of the surface areas, image analysis was performed on a personal computer using the public domain NIH Image program (Image J1.36b). For FACS analysis, each assay was repeated 3 times. Statistical analysis was performed using the Student t test.

Results

Donor MHC+ Cells Migrate Systemically to Host Lymphoid Organs.

First, migration kinetics of donor cells was examined. A considerable number of donor MHCI+ cells and MHCII+ cells were present in the T cell areas and B cell areas of the host spleen (Fig. 1A,B), LNs, and Peyer's patches 6-12 hours after liver transplantation. The migration pattern of donor MHCI+ and MHCII+ cells in BMC transfer (Fig. 1C) and TDL transfer groups were basically same as the liver transplant group. Because the TDL transfer group contained both T cells and B cells,12 we considered the round MHCI+ cells in the T cell areas and the MHCII+ cells in the B cell areas to be primarily donor T cells and B cells, respectively.

Figure 1.

Whole view of spleen 1 day after (A,B) liver transplantation or (C) BMC transfer. Double immunostaining for (A) donor MHCI (blue) and immunoglobulin M (brown), (B) donor MHCII (blue) and TCRαβ (brown), and (C) donor MHCI (blue) and type IV collagen (brown). (A) Donor MHCI+ cells scattered in the T cell area (P) and B cell area (lymph follicle, F). (B) Donor MHCII+ cells are many in the B cell area and a few in the T cell area. Inset is a higher magnification of arrowed area showing 2 donor MHCII+ cells with dendritic shape clustering with host T cells. (C) Even 2.5 × 108 bone marrow cells (blue) showed less intense migration than for the liver transplant group.

Of note, we found a small but significant number of donor MHCII+ cells with a dendritic shape forming clusters with host T cells in the T cell areas of the spleen (Fig. 1B, inset), LNs, and Peyer's patches in liver transplant and BMC transfer groups. In contrast, in the TDL transfer group, there were very few cluster-forming donor MHCII+ cells in the T cell areas.

The number of donor MHCI+ and MHCII+ cells peaked at approximately 1-2 days and decreased after day 3; the cells had almost disappeared by day 4. By FACS estimation, the number of migrated donor MHCI+ cells in the host spleen 2 days after liver transplantation, BMC transfer, and TDL transfer (Fig. 2A) was approximately 2 × 106, 1 × 106, and 7 × 106, respectively. The Peyer's patches of these 3 groups yielded approximately 1.5 × 104, 1 × 104, and 5 × 104 donor MHCI+ cells, respectively (Fig. 2B). Because the relative cell number per 108 injected cells was calculated for the BMC and TDL groups, the BMC transfer group contained many fewer migrating cells than the TDL group. Thus, major migrants were lymphocytes in all 3 groups, and donor MHCII+ DC-like cells were found in the liver transplant and BMC-transferred groups.

Figure 2.

FACS analysis showing migration of a donor DC population 2 days after liver transplantation (LTx), bone marrow cell (BMC) transfer, or thoracic duct lymph cell (TDL) transfer. Number of migrated donor MHCI+ cells in (A) host spleen and (B) Peyer's patches. For BMC and TDL groups, the relative cell number per 108 injected cells is shown (n = 3-4). (C) Phenotype of migrated donor MHCII+ cells in LTx group, indicating a presence of CD11c+ and CD103+ cells (dotted squares). Numbers indicate percentages of CD11c+ and CD103+ cells/total cells. (D) Proportion of CD11c+ and CD103+ DCs among donor MHCII+ cells in host Peyer's patches. Control, normal mouse IgG. n = 3, *P < 0.05, **P < 0.005.

A Donor DC Population Migrates to Host Secondary Lymphoid Organs.

To know whether the migrated MHCII+ donor cells contained DCs, host spleen and Peyer's patches were examined with FACS. Because rat DCs are CD11c+,8 and at least partly CD103+,15 mAbs to these antigens were used. Although intraepithelial T cells are CD103+, they are MHCII,6 and can be excluded from the MHCII+ cell fraction.

Donor MHCII+ cells in the spleen and Peyer's patches 2 days after liver transplantation included CD45R+ B cells and a few CD3+ T cells (Fig. 2C). In addition, donor MHCII+ cells expressing either CD11c or CD103 were readily detected in the spleen (Fig. 2C) and Peyer's patches (Fig. 2C,D) and were phenotypically defined as conventional DCs. Surprisingly, in the Peyer's patch suspensions, up to 8%-9% of donor MHCII+ cells were CD11c+ or CD103+. FACS analysis of liver nonparenchymal cells of donor ACI rats demonstrated the presence of cells with a phenotype similar to the migrated population (Fig. 3A). Concerning costimulatory molecule and homing receptors, CD103+ DCs in the low-density fraction were mostly CD86 (Fig. 3B), CD62L, but CD49d+ (Fig. 3C,D).

Figure 3.

Presence and phenotype of DCs in donor liver, BMCs, and TDLs. (A) Low and high buoyant density liver nonparenchymal cells (NPCs) were stained for MHCII and mAbs. Numbers indicate percentages of mAb+ cells/total cells. (B) Almost negligible expression of CD86 by CD103+ liver DCs. (C) MHCIIhighCD3IgCD163 (non-T, non-B, and non-macrophage) liver putative DCs (boxed area) were mostly positive for CD103 but negative for CD62L. (D) MHCII+CD103+ liver DCs were negative for CD62L but positive for MAdCAM-1 ligand, CD49d (integrin α4 chain). (E) Percentage of DCs among MHCII+ cells from freshly prepared BMCs or TDLs. Control, normal mouse IgG.

In the BMC transfer group, host Peyer's patches (Fig. 2D) and donor cell suspensions (Fig. 3C) similarly contained donor DCs. In contrast, in the TDL transfer group, significantly fewer donor DCs were detected in the Peyer's patches (Fig. 2D) and TDL suspensions (Fig. 3E). Thus, donor MHCII+CD11c+ and/or CD103+ DCs did migrate to host lymphoid organs after liver transplantation or BMC transfer, and CD103+ DCs in the donor liver were CD86CD62LCD49d+.

Liver Transplantation Induces a Systemic Host T Cell Proliferative Response.

To investigate a presence of allosensitization in host lymphoid organs, the proliferative response was examined. Beginning on day 2 after liver transplantation, not only spleen but also LNs and Peyer's patches of host rats demonstrated a vigorous proliferative response in the T cell area (Fig. 4). Most BrdU+ cells were TCR+ (Fig. 5A), indicating a host T cell response. More detailed analysis of the splenic T cell area showed that a significant proliferative response began 36 hours after liver transplantation, and CD8β+ cells were actively expanding (see below). The results indicate that allosensitization occurs systemically in secondary host lymphoid organs from 36-48 hours after liver transplantation.

Figure 4.

Kinetics of the proliferative response in host lymphoid organs after liver transplantation. A significant proliferative response was observed in all organs starting on day 2. No significant response was observed 2 days after syngeneic liver transplantation. n = 3, *P < 0.05, **P < 0.01.

Figure 5.

Cluster formation between donor DCs and proliferating host T cells. (A) Triple immunostaining of spleen for TCRαβ (blue), type IV collagen (brown), and BrdU (red) 2 days after liver transplantation. Proliferating cells (red) are mostly TCR+ (arrowheads). Triple immunostaining of (B) spleen and (C) Peyer's patches 1 and 2 days after liver transplantation, respectively: donor MHCII (blue), type IV collagen (brown), and BrdU (red). Clusters of donor MHCII+ cells and proliferating cells are present in T cell areas (arrows). (B, inset) Higher magnification of a typical cluster. Green triangle indicates T cell area of Peyer's patches (interfollicular area). (D-G) Triple immunostaining of Peyer's patch serial sections shows that donor MHCII+ cell (blue) (D, arrow) clustering with BrdU+ cells (red) is CD11c+ (blue) (E, arrow) and that donor MHCII+ cells (blue) (F, arrows) clustering with BrdU+ cells (red) are CD86+ (blue) (G, arrows). Abbreviations: F, lymph follicle; P, splenic T cell area. *High endothelial venule.

Donor DCs and Proliferating Host T Cells Form Clusters.

To know whether migrated donor DCs form the BrdU+ clusters or not, immunohistological analysis was performed. On day 1 to 2 after liver transplantation, T cell areas of host spleen (Fig. 5B), LNs, and Peyer's patches (Fig. 5C) contained a small but significant number of donor MHCII+ cells clustering with BrdU+ cells. Regarding the phenotype of the BrdU+ cluster-forming donor MHCII+ cells in the T cell area of Peyer's patches (Table 2), approximately 70% were CD11c+ (Fig. 5D,E) or CD86+ (Fig. 5F,G) 2 days after liver transplantation. In the BMC transfer group, most cluster-forming cells in the Peyer's patches were also CD11c+. In contrast, the number of cluster-forming cells in the Peyer's patches of the TDL transfer group was less than one-tenth of that in the liver transplant group (Table 2). In addition, a few cluster-forming cells in the spleen of the liver transplant group were TCR+, showing that migrated MHCII+ T cells can form BrdU+ clusters with host proliferating T cells (data not shown). The significance is unclear but these clusters may be sites for killing of donor cells by activated host cells.4

Table 2. Donor MHCII+ Cluster-Forming Cells in Interfollicular Area of Peyer's Patches
 LTxBMCTDL
  • Two days after liver transplantation (LTx), bone marrow cell (BMC) transfer or thoracic duct lymph cell (TDL) transfer.

  • *

    Proportion of CD11c+ or CD86+ donor MHCII+ cells / total donor MHCII+ cluster forming cells that contain two or more BrdU+ cells. Mean ± SD (n = 3).

  • 22 cells out of 33 cells are clearly host MHCI-negative.

No./ interfollicular area1.10 ± 0.380.73 ± 0.010.10 ± 0.01
% CD11c+ donor MHCII+*66.7 ± 13.263.1 ± 8.9nil
% CD86+ donor MHCII+*70.0 ± 12.0NTNT
No. of host MHCI donor MHCII+/total donor MHCII+22/33NTNT

In spleen cross-sections, BrdU+ clusters containing 2 or more BrdU+ cells/T cell area were first observed at 24 hours, and the number of clusters peaked at 48 hours (Fig. 6B). On the other hand, the number of BrdU+ cells increased slightly but not significantly at 24 hours (Fig. 6A). A significant proliferative response began 36 hours after liver transplantation both in the splenic T cell area (Fig. 6A) and the nonsinusoidal area of graft liver (Fig. 6E). Thus, the BrdU+ clusters appeared 12 hours before a significant host T cell proliferative response. Early cluster formation also occurred in the LNs and Peyer's patches (data not shown). In the Peyer's patches, at 2 days after liver transplantation and BMC transfer, the number of BrdU+ clusters was approximately 1.1 and 0.7 per interfollicular area (triangular T cell area, Fig. 5C), respectively (Table 2). Together, donor CD11c+ DCs formed BrdU+ clusters where these DCs became CD86+ and host alloresponse originated.

Figure 6.

Kinetics of the proliferative response and cluster formation in host spleen after liver transplantation. (A) Number of BrdU+ cells/mm2 of host splenic T cell area. *P < 0.01 versus control; NS, not significant. (B) Number of cluster-forming donor MHCII+ cells containing one or two or more BrdU+ cells in the T cell area per spleen cross-section. (C) Number of donor proliferating cells in the T cell area per spleen cross-section. (D) Ratio of number of BrdU+ donor cells in host DC clusters to total number of BrdU+ donor cells. (E) Number of BrdU+ cells/mm2 of nonsinusoidal area of graft liver. n = 3.

Host DCs and Proliferating Cells Form Clusters.

Next, the role of host resident DCs was investigated. Some donor MHCI+ cells also exhibited active proliferation. BrdU+ donor MHCI+ cells appeared in the T cell area 24 hours after liver transplantation (Figs. 6C and 7A). More than 80% of these cells were closely associated with host MHCII+ cells (Figs. 6D and 7A). Because dendritically shaped host MHCII+ cells are mostly resident interdigitating DCs,4 and donor MHCI+ cells in the T cell area were mostly TCR+ (data not shown), the association between host MHCII+ cells and donor MHCI+ represents clusters of host resident DCs and donor T cells. This indicates a direct graft-versus-host reaction (GvHR), where host resident DCs allosensitize migrated donor T cells. In addition, we observed diffuse clustering of host BrdU+ cells (donor MHCI) and host MHCII+ cells in the T cell area starting on day 2 (Fig. 7B). Furthermore, donor MHCI+ fragments were detected within host MHCII+ cells (Fig. 7B, inset). This suggests phagocytosis and processing of donor MHC antigens by host resident DCs and the indirect pathway whereby host resident DCs present donor MHC antigens to host T cells. In this respect, some of the donor MHCII+ cluster-forming cells might also be host DCs expressing donor MHCII on their surfaces, because donor DC molecules may be acquired by host DCs and transfer of intact MHC class II via exosomes16 or similar mechanisms may occur. Accordingly, we examined their phenotype. The fact that 22 donor MHCII+ cluster-forming cells out of 33 were clearly host MHCI-negative (Table 2) indicates that most of the donor MHCII+ cluster-forming cells were not host-derived. The two types of BrdU+ clusters and donor MHC-antigen uptake by host resident DCs were also observed in the BMC and TDL transfer groups (data not shown.)

Figure 7.

Cluster formation by host DCs and donor or host proliferating cells in host spleen. (A) One day after liver transplantation, host DCs (host MHCII+, brown) cluster (arrows) with donor proliferating cells (donor MHCI+, blue, with BrdU+ nucleus, red). (B) Two days after transplantation, host DCs (brown) cluster (arrows) with host proliferating cells (BrdU+, red). (B, inset) Ingestion of donor MHCI+ fragments (red dots, arrowhead) by host DCs (green).

Liver Transplant and Bone Marrow Transfer Induce a Strong CD8+ T Cell Response.

Finally we compared CD8+ T cell responses among the three groups. Host spleens from the liver transplant (Fig. 8A) and BMC transfer groups demonstrated an active CD8+ T cell proliferative response (Fig. 8D,E) on days 1-4. Up to 600 BrdU+ cells/mm2 were clearly CD8β+ in the splenic T cell area on day 2 after transplantation. In the Peyer's patches of the liver transplant group, donor MHCII+ cell clusters with CD8β+BrdU+ cells and CD8βBrdU+ cells were found (Fig. 8C). In contrast, in the TDL transfer group, although there was a comparable proliferative response in the T cell area (Fig. 8D), the CD8+ response was significantly weaker than for the other groups on days 2 and 3 (Fig. 8B,E). Thus, migrated donor DCs induced a strong CD8+ T cell proliferative response most probably in the BrdU+ cluster.

Figure 8.

CD8 response in (A,B,D,E) host spleens and (C) Peyer's patches after (A,C) liver transplant (LTx), BMC transfer, or (B) TDL transfer. (A-C) Triple immunostaining on day 2 for CD8β (blue), type IV collagen (A,B, brown) or donor MHCII (C, brown) and BrdU (red). Many CD8β+BrdU+ cells (arrowheads) are found in (A) the LTx group but fewer in (B) the TDL group. In Peyer's patches, CD8β+BrdU+ or CD8βBrdU+ cells cluster with donor MHCII+ cell (C, brown, arrow). (D,E) Number of total BrdU+ cells (D) and CD8β+BrdU+ cells (E) per mm2 of T cell area of spleen. *P < 0.05 versus TDL group. n = 3.

Discussion

The present study demonstrates that rat liver allotransplantation induces a systemic blood-borne transmigration of donor MHCI+ and MHCII+ cells from the graft liver to the host secondary lymphoid organs and an intense proliferative response in host CD8+ T cells in these regions. FACS analysis revealed that the migrated donor cells included conventional DCs as well as T cells and B cells. The BMC transfer group but not the TDL transfer group also contained migrating DCs and induced CD8 response.

Upon immunohistologic analysis, we found a small but significant number of donor DCs forming BrdU+ clusters with proliferating host T cells in the T cell areas of the spleen and Peyer's patches. T cell proliferative responses originated within these clusters (Fig. 7), representing sites of intrahost direct allosensitization by migrated donor DCs, in other words, a direct host-versus-graft reaction (HvGR). Notably, this direct HvGR was also observed in the BMC transfer group, but not in the TDL transfer group, which lacked DCs. Previous studies reported the migration of donor MHCII+CD11c+ cells8 and cluster formation between donor MHCII+ cells and proliferating cells in the spleen after rat liver transplantation,7 but this is the first report of the systemic transmigration of a donor DC population to the host secondary lymphoid organs, the kinetics of BrdU+ clusters, and the presence of intrahost direct allosensitization.

Because the Peyer's patches have no afferent lymphatics, blood vessels are the only routes of entry for migrating cells. A likely site for cell entry is the high endothelial venule, because these vessels predominate in the T cell area in which cluster-forming donor cells were found (Fig. 5C-E). CD103+ DCs in the low-density fraction were CD62L but expressed CD49d (integrin α4 chain). Because CD49d is a homing ligand of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expressed on the high endothelial venule of the Peyer's patches, the presence of CD49d on donor DCs may facilitate their migration to the Peyer's patches. Therefore, our results demonstrate the presence of a unique DC population in the liver and bone marrow that can transmigrate through the blood vessel wall, probably the high endothelial venule, of peripheral lymphoid organs.

DCs must be relatively immature to accomplish this migration. It is thought that in the steady state, only DC precursors can enter peripheral organs via the blood. DC progenitors in mouse liver17 and bone marrow9 reportedly have similar migratory capability. In contrast, semimature DCs at the antigen-transporting stage do not transmigrate through the high endothelial venule.6, 18, 19 However, recent reports indicate that some DCs that carry antigens administered locally may be able to disseminate systemically to the peripheral LNs20 and suggest that blood-borne antigen transport from the periphery to lymphoid organs may occur.21 In this study, DCs in the donor liver were mostly immature CD86 cells. However, the migrated DCs quickly clustered with host T cells and acquired a mature phenotype, CD86+,9 and the CD8+ T cell proliferative response originated in these clusters within 24 hours after liver transplantation. This suggests that the migrating DCs were semimature and could quickly mature after transmigration. Alternatively, we cannot ignore the possibility that some of them might be derived from much earlier precursors that behave similarly. Together, we conclude that migrating DCs are an immature population that can transmigrate through the blood vessel wall and quickly mature at migrated sites. They may undergo maturation through interactions with vascular endothelial cells and T cells.22

In all three groups, we observed a transitory direct GvHR by migrated donor T cells in host lymphoid organs. These cells may become GvH effector cells, because liver allografts can induce GvH disease in immunocompromised hosts.23 However, host rats were not immunosuppressed in our study, and the GvHR may be temporary due to suppression by activated host cells. Our data also suggest the occurrence of the indirect pathway by host DCs—in other words, indirect HvGR—in all three groups. In the TDL transfer group, in particular, this indirect HvGR is likely to be mostly responsible for host T cell activation. Together, the systemic T cell proliferative responses were induced by two intrahost direct pathways, the HvGR and the GvHR. Probably, indirect HvGR was operative in host lymphoid organs following liver transplantation and transfer of BMCs. In contrast, indirect HvGR and direct GvHR, but not direct HvGR, were induced in the TDL transfer group.

Host spleens of the liver transplant and BMC transfer groups, but not the TDL transfer group, showed an active CD8+ T cell proliferative response (Fig. 8). The presence of migrating donor DCs and the direct HvGR seem to be central to this difference. Our results demonstrate that donor DCs can induce a much stronger host CD8 response than can donor non-DC leukocytes. Conversely, host DCs must also induce a CD8 response from donor T cells during the transitory direct GvHR; this is probably indicated by the weak but significant CD8 response in all three groups on day 1 (Fig. 8). It has been suggested that DC progenitors in the mouse liver poorly stimulate allogeneic T cells and selectively induce a Th2 response when transferred in vivo.24 This may reflect a species difference, because mice spontaneously accept allogeneic liver transplants without immunosuppression.2

Although it is widely believed that direct allosensitization by donor DCs is responsible for acute rejection,5, 25 the indirect pathway by host DCs might also be involved.8, 26 Bowen et al.11 suggested that the outcome of intrahepatic CD8+ T cell responses might be determined by whether primary activation occurs within secondary lymphoid organs or the liver. Using a transgenic mouse model in which antigen is expressed in both the liver and LNs, they showed that naive CD8+ T cells activated within the LNs could mediate hepatitis, whereas cells sensitized within the liver exhibited defective cytotoxic function and a shortened half-life and did not mediate hepatocellular injury. This suggests that irrespective of direct or indirect pathways, a CD8 response via intrahost allosensitization, but not intrahepatic allosensitization, is crucial for acute rejection of a liver graft. Because in this study, transmigrating donor DCs induced a systemic CD8 response in the host lymphoid organs, we consider intrahost direct allosensitization by donor DCs to be mainly responsible for acute rejection of rat liver transplants. If this theory were correct, inhibition of donor DC migration would suppress acute rejection. This hypothesis is now under study.

In conclusion, we have demonstrated an immature DC population in the liver as well as bone marrow that can transmigrate to the secondary lymphoid organs, can quickly mature, and induces allosensitization systemically. This population might serve as an alternative vector for intravenous delivery in antigen-pulsed DC immunotherapy, which is currently problematic due to the difficulty of delivering a sufficient quantity of vector to the secondary lymphoid organs via the blood.19 Further investigation of transmigrating DCs would facilitate clinical intervention to control both the alloresponse in the initial stage of transplantation and DC immunotherapy.

Acknowledgements

We are grateful to Drs. Ralph Steinman, Yong-Jun Liu, Hiromitsu Kimura, Koji Matsushima, Hiroyuki Yoneyama, Kazuhito Rokutan, and Takamasa Ueno for valuable discussion and suggestions and to Junko Sakumoto, Kyoko Ohta-Kato, and Yasuko Nonaka for their excellent technical support.

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