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(Journal of Leukocyte Biology. 2001;69:705-712.)
© 2001 by Society for Leukocyte Biology

In vivo roles of donor and host dendritic cells in allogeneic immune response: cluster formation with host proliferating T cells

Takahito Saiki*,{dagger}, Taichi Ezaki*, Michio Ogawa{dagger}, Keiko Maeda{ddagger}, Hideo Yagita§ and Kenjiro Matsuno*

Departments of
* Anatomy II and
{dagger} Surgery II, Kumamoto University School of Medicine, Kumamoto 860-0811, and
{ddagger} Atopy Research Institute and
§ Department of Immunology, Juntendo University School of Medicine, Tokyo 113-0033, Japan

Correspondence: Kenjiro Matsuno, M.D., Department of Anatomy (Macro), Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan. E-mail: kenjiro{at}dokkyomed.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Possible roles of dendritic cells (DCs) in allogeneic immune responses in host lymphoid tissues were characterized in situ by using rat DC transfer and cardiac transplantation models. When allogeneic DCs were intravenously injected, these cells selectively migrated to the T-cell area of hepatic lymph nodes, with peak accumulation at 18 h after injection. Donor DCs and proliferating host T cells formed clusters (rosettes) in which the T-cell proliferative response started. The donor DCs were CD80+ CD86+ and, ultrastructurally, were in intimate contact with lymphoblasts within the rosettes. As a novel finding, some of the migrated donor DCs were quickly phagocytosed by putative host interdigitating DCs. By 48 h, the remaining donor DCs had disintegrated within the rosettes. Host interdigitating DCs also formed rosettes throughout the T-cell area, and their kinetics correlated well with that of the T-cell proliferation. In the cardiac allograft model, a few donor DCs selectively migrated to the host spleen and hepatic nodes. Rosette formation by donor and host DCs, phagocytosis of donor DCs, and the T-cell proliferative response occurred in much the same fashion as they did in the first experiment. We conclude that the donor rosettes at the early stage represent the sites of direct allosensitization and those at the late stage represent donor-DC killing. Host rosettes are the sites of T-cell proliferation. In this structure, phagocytosed donor-DC-derived antigens are presumably indirectly presented.

Key Words: dendritic-cell migration • cardiac transplantation • hepatic lymph nodes • interdigitating dendritic cells • allosensitization • rat


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The dendritic cell (DC) is a key initiator of allogeneic immune responses during organ transplantation and, therefore, is critical for graft rejection [1 ]. 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 sensitization). Host DCs may take up graft MHC antigens and present them to host T cells (indirect sensitization or cross-priming). The roles of donor and/or host DCs in host T-cell sensitization as well as in the consequent immune responses within host lymphoid tissues, however, still need to be elucidated [2 3 4 ].

Previously, we found that intravenously (i.v.) injected allogeneic rat DCs translocated from the hepatic sinusoids to the lymphatic system and eventually accumulated in the T-cell area (paracortex) of the draining hepatic lymph nodes (LNs) [5 , 6 ]. Migrated DCs clustered with host T cells, and an allospecific T-cell proliferative response occurred in these LNs, suggestive of the direct sensitization pathway. Similarly, in a rat cardiac allotransplantation model [7 ], not only donor DCs but also host DCs which had been recruited to the graft executed blood-borne migration to the host hepatic LNs and spleen. In these tissues, effector cells were predominantly produced, suggesting that these migrated DCs might be responsible for the sensitization. Clustering of migrated DCs with host T cells may be indicative of the occurrence of some sensitization processes. However, the clustering does not in itself conclusively indicate the site of sensitization, since DCs can nonspecifically cluster with T cells [8 , 9 ] and B cells [10 ]. Besides, migrated DC-derived antigens might be processed and presented by host-resident DCs [interdigitating DCs (IDCs)] via an indirect pathway [4 ]. Thus, the significance of DC–T-cell clustering in situ still needs to be elucidated.

In the present study, we analyzed in situ allogeneic immune responses, especially those involved in the process of sensitization by DCs in host lymphoid tissues, by double- or triple-immunostaining techniques. Time kinetics for mutual relationships among donor DCs, host DCs, and host proliferating cells were examined in both an adoptive DC transfer model and a rat cardiac transplantation model. The former model was used to reveal an effect of the migrated donor DCs alone since other parameters, such as cytokines or soluble donor MHC antigens in the blood, could be neglected. The sensitization process in rat cardiac transplantation was also analyzed. We found a preferential clustering of both donor and host DCs with host proliferating T cells, which were crucial for the allogeneic immune responses, including the sensitization process.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Inbred male DA (RT1AaBa) and Lewis (RT1AlBl) rats were supplied by the Laboratory Animal Center for Experimental Research (Kumamoto University School of Medicine, Japan). They were reared under specific-pathogen-free conditions. Handling and care of animals were under the local regulation of the Laboratory Animal Center for Experimental Research, Kumamoto University.

Isolation and adoptive transfer of lymph DCs
The celiac and mesenteric LNs were surgically removed, which resulted in a direct influx of peripheral hepatointestinal lymph into the thoracic duct after regeneration of lymphatic vessels [11 ]. Thus, DCs in the hepatointestinal lymph could be directly obtained by cannulating the thoracic duct after 6 weeks. Rats were i.v. injected with 100 µg of Escherichia coli-derived lipopolysaccharide (Sigma Chemical Co., St. Louis, MO) to stimulate an increase in DC output [12 ]. DCs in the thoracic-duct lymph were enriched by 16% Nycodenz (Nycomed Pharma, Oslo, Norway) gradient centrifugation [13 ]. The DC preparation was 60–80% pure and had a viability of >95%. Paramagnetic-latex-laden DCs were also isolated from the hepatic lymph [5 ]. The purity of latex-laden DCs was 80–90%, with a viability >90%. Isolated DCs in the hepatointestinal lymph or purified latex-laden DCs from DA rats (106 cells) were i.v. injected into recipient Lewis rats. At 6, 18, and 24 h and at 2, 3, and 4 days after the cell transfer, the hepatic LNs of host rats were excised and fresh cryosections were prepared. Proliferating cells were labeled by i.v. injecting bromodeoxyuridine (BrdU; Sigma Chemical Co.; 2 mg/100 g of body weight) 1 h prior to sacrifice.

Heart transplantation
Heterotopic cardiac transplantation from donor DA rats to the necks of host Lewis rats was performed (7). In this model, Lewis host rats rejected DA hearts about 7 days after transplantation (mean survival time: 6.67 ± 0.58 days; n = 6). At 1, 2, 3, 4, and 5 days after transplantation, host rats received BrdU and were sacrificed 1 h later. Grafted hearts and host spleens and hepatic LNs were excised, and fresh cryosections were prepared.

Regrafting of transplanted hearts to secondary hosts was performed as described previously [7 ]. DA rat hearts were first grafted to Lewis rats and then regrafted to normal DA rats 3 days after the first transplantation. Thirty-six hours after the regraft operation, the secondary hosts received BrdU, and they were sacrificed 1 h later.

Antibodies and reagents
Mouse monoclonal antibodies (mAbs) specific for rat determinants, including antibodies against CD5 (OX19), CD8 (OX8), a pan-B-cell marker (HIS24), polymorphic MHC class I (anti-RT1Aa, MN4-91-6) and class II (anti-RT1Ba/c, OX76) antigens of the DA rat, and MHC class II antigens of the Lewis rat (anti-RT1Bl, OX3) were obtained from Serotec Ltd. (Kidlington, Oxford, UK). A mAb against activated macrophages (antisialoadhesin; also known as TRPM3) was obtained from BMA Biomedicals AG (Augst, Switzerland). A mAb against BrdU was purchased from Novocastra Laboratories Ltd. (Newcastle upon Tyne, UK). Mouse mAbs to rat CD80 (clone 3H5) and CD86 (clone 24F) were produced and characterized [14 ]. A rabbit polyclonal antibody against mouse type IV collagen, used to outline tissue framework [15 ], was purchased from Cosmo Bio (Tokyo, Japan). As secondary antibodies, an alkaline phosphatase (ALP)-labeled goat immunoglobulin (Ig) (A9316; Sigma Chemical Co.), an ALP-labeled sheep F(ab')2 (A4812; Sigma) to mouse Ig, a horseradish peroxidase (HRP)-labeled rabbit Ig to mouse Ig (P161; Dako Corp., Santa Barbara, CA), and an HRP-labeled goat F(ab')2 to rabbit Ig (55693; Cappel, Aurora, OH) were employed.

Immunohistological procedures
The double- and triple-immunostaining methods used in this study were described previously [5 , 16 ]. In brief, fresh 4–8-µm-thick cryosections were double immunostained for mAb(s) by the repetition of either the indirect immunoperoxidase technique with a diaminobenzidine substrate (brown) or the immuno-ALP technique with a Vector Blue substrate kit (Vector Laboratories Inc., Burlingame, CA). Additional BrdU staining was performed by the indirect immuno-ALP technique with a Vector Red substrate kit (Vector Laboratories Inc.).

Immunoelectron microscopy
Lymph DCs isolated from DA rats (106 cells/mL) were i.v. injected into Lewis rats. At 24 h postinjection, hepatic LNs were fixed by perfusion with periodate-lysine-paraformaldehyde. Small blocks were frozen, and 50-µm-thick cryosections were immunoreacted with a mAb to MHC class I of the DA rat (MN4-91-6). An HRP-conjugated sheep F(ab')2 anti-mouse Ig antibody (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) was used as a secondary antibody for the indirect enzyme immunostaining. The immunoreactions were developed with diaminobenzidine. Sections were postfixed with 1% osmium tetroxide, electron-stained with 3% uranyl acetate, and embedded in an Epon-Araldite mixture. Ultrathin sections (about 70 nm thick) were examined at 80 kV with a JEOL 100CX electron microscope (Nihon Densi, Tokyo, Japan).

Experimental design
In the first adoptive DC transfer experiment, the donor DCs were readily identified as donor-type MHC I+ or MHC II+ cells. The hepatic LNs of host Lewis rats were triple immunostained for either donor MHC I or MHC II (MN4-91-6 or OX76; ALP, blue), BrdU (ALP, red), and type IV collagen (HRP, brown). The host DCs were identified as host-type (polymorphic) MHC II-single-positive cells without macrophage and lymphocyte markers. To identify the host DCs and proliferating cells, the samples were triple immunostained for macrophages, B cells, and T cells (a cocktail of TRPM3, HIS24, and OX19; HRP, brown); host MHC II antigen (OX3; ALP, blue); and BrdU (ALP, red). To examine the relationship between donor DCs and host MHC II+ cells in the hepatic LNs, cryosections were double immunostained for donor MHC II (OX76; HRP, brown) and host MHC II (OX3; ALP, blue) antigens. The samples containing latex-laden DCs were also double immunostained for CD80 or CD86 (ALP, blue) and host MHC II antigen (OX3; HRP, brown) to reveal expression of costimulatory molecules. As described later, both donor and host DCs formed cell clusters containing BrdU+ cells. We defined this cluster as a donor- or host-DC rosette (hereafter termed donor/host rosette). The kinetics of formation of rosettes by either donor or host DCs was analyzed by counting the number of rosettes which contained either one BrdU+ cell or two or more BrdU+ cells. The ratio of the number of donor DCs forming the rosette to the total number of donor DCs was also calculated. For host DCs, the number of BrdU+ cells either inside or outside of the rosette was counted and the ratio of the number of BrdU+ cells within the rosette to the total number of BrdU+ cells was estimated as well. Immunoelectron microscopy was performed to reveal a direct contact of donor DCs with host T cells within the rosettes.

In the second experiment, using the rat allogeneic cardiac transplantation model, cryosections of the host spleens were immunostained and quantified in a fashion similar to that of the first experiment. Donor DCs were identified as donor MHC II+ cells because migrated donor cells were considered to be primarily DCs [7 ]. For quantitative analyses of host rosettes, the mAb to T cells (OX19) was omitted from the first staining to negatively depict the T-cell area [the periarterial lymphoid sheath (PALS)] in the white pulp. This staining resulted in a distribution of host MHC II-single-positive cells in the PALS similar to that obtained in the original staining in which OX19 was added (data not shown). The spleens of secondary hosts in the regraft experiment as well as the grafted hearts were also examined. Three or four rats were used for each experiment. Each parameter was measured in a blinded fashion and expressed as the mean ± SD. A statistical analysis was performed using Student’s t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fate of transferred DCs
As reported previously [5 ], i.v.-injected rat DCs selectively migrated to the T-cell area (paracortex) of hepatic LNs (Fig. 1a ). These cells appeared in the hepatic LNs as early as 6 h postinjection (data not shown), with peak accumulation at 18 h (Fig. 2 ), and then disintegrated; they had almost disappeared by 48 h after injection. From 18 h after injection, donor MHC II+ "dotty" fragments were observed in host MHC II+ cells with DC morphology in the paracortex (Fig. 2) . The location and morphology of these host cells were similar to those of the host DCs, identified as host MHC II-single-positive cells by triple immunostaining (see below). In addition, there were a few macrophage populations in the paracortex of rat LNs [17 , 18 ]; these cells were considered to be primarily host IDCs. At day 2, the remaining donor DCs appeared to have disintegrated and exhibited weak granular MHC II staining.



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  		Figure 1. Kinetics of donor DCs, host proliferative response, and rosette formation in hepatic LNs after i.v. transfer of DCs. Values are means ± SD (n = 3) of data for 6-µm-thick sections. (a) Absolute number of migrated donor DCs, identified as donor MHC I+ or MHC II+ cells, in the mid-cross sections. (b) Proliferative response in the paracortex, expressed as the number of BrdU+ cells per square-millimeter section. (c) Absolute number of donor rosettes containing either one BrdU+ cell or two or more BrdU+ cells in the mid-cross sections. (d) Ratio of the number of donor DCs forming the rosette to the total number of donor DCs. (e) Number of host rosettes containing either one BrdU+ cells or two or more BrdU+ cells per square-millimeter section. (f) Ratio of the number of BrdU+ cells within the rosette to the total number of BrdU+ cells.

 


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  		Figure 2. Accumulation of i.v.-injected donor DCs (brown) in the T-cell area of hepatic LNs at 18 h postinjection, as determined by double immunostaining for donor MHC II (brown) and host MHC II (blue). (a) Donor DCs (brown) and host MHC IIhigh+ cells (blue) with polygonal or dendritic shape, mostly host IDCs, and donor DCs intermingle in the T-cell area [paracortex (P)]. F, B-cell follicle. Magnification, x125. (b) A higher magnification of the T-cell area. Note donor MHC II+ dotty fragments are within host MHC II+ cells (arrowheads), indicative of phagocytosis of donor DCs by host putative IDCs. One donor DC (arrow) looks intact. Magnification, x500.

 
Rosette formation by donor DCs after adoptive DC transfer
Allogeneic DCs (106) induced a significant T-cell proliferative response in the host hepatic LNs, with a peak at day 2 after injection (Fig. 1b) . This response is antigen-specific and donor DC-dependent, since the same number of either syngeneic DCs or allogeneic unseparated cells did not induce a proliferative response, as described previously [5 ]. Donor DCs formed clear cell clusters with BrdU+ host cells (Fig. 3a ; donor rosettes), which were mostly T cells (data not shown). The donor rosettes containing two or more BrdU+ cells were first observed 18 h after DC transfer; their number peaked at 24 h and had quickly fallen by 48 h (Fig. 1c) . Notably, at 48 h, when donor DCs were disintegrating and almost disappearing, the ratio of the number of donor DCs forming the rosette to the total number of donor DCs had relatively increased (Fig. 1d) . In other words, most of the donor DCs remaining within the rosette were degenerating. At 18 h, BrdU+ cells were rather confined in and around the rosette, but they became scattered throughout the T-cell area of hepatic LNs from 24 h after injection (Fig. 3b) . The majority of donor DCs that had accumulated in the hepatic LNs, as evidenced by the presence of latex particles in their cytoplasm and by their lack of host MHC II antigen, expressed both CD86 (Fig. 4 ) and CD80 (data not shown). Donor DCs within the rosettes had intimate contact with surrounding cells, which were primarily activated lymphoblasts, as shown by immunoelectron microscopy (Fig. 5 ).



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  		Figure 3. Formation of clusters by donor DCs and BrdU+ host cells in the T-cell area of hepatic LNs at 24 h postinjection, determined by triple immunostaining for donor MHC I (blue), BrdU (red), and type IV collagen (brown). (a) Two donor DCs (blue) are surrounded by host BrdU+ cells (red nuclei), forming typical rosettes. Magnification, x500. (b) BrdU+ cells scatter diffusely throughout the paracortex (P) of the hepatic LNs. Magnification, x250.

 


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  		Figure 4. A majority of donor DCs, identified by the presence of ingested latex particles (brown aggregates) in their cytoplasm and by their negativity for host MHC II (brown), in the hepatic LNs are CD86+ (blue, arrows). Note that some host MHC II+ cells are also CD86+ (bluish brown, arrowheads). Magnification, x500.

 


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  		Figure 5. Immuno-electron micrograph of the donor DC rosette. Twenty-four hours after i.v. injection into allogeneic host, a DC (D) migrated into the paracortex of hepatic LNs and formed an intimate contact with surrounding lymphocytes, some of which were lymphoblasts (asterisks). The donor DCs with dendritic processes were detected by immunostaining for donor MHC I. Magnification, x5,700.

 
Rosette formation by host DCs after adoptive DC transfer
Host IDCs could be readily identified as host-type MHC II-single-positive DCs without B-cell, T-cell, and macrophage markers (Fig. 6 ). Most host MHC II-single-positive cells in the T-cell area were large and had MHC II+ dendritic processes, while other MHC II+ cells, such as B cells and macrophages, were blue-brown because of the first diaminobenzidine staining and could be easily discriminated. The morphology and location of host MHC II+ cells containing donor MHC II+ dotty fragments (Fig. 2b) were similar to those of these IDCs. Host IDCs constantly formed rosettes even during steady state, but the number of host rosettes had increased by 24 h after donor DC injection and reached its peak on day 1 or 2 (Fig. 1e and 6) . This kinetics correlated well with that of BrdU+ cells in the T-cell area, i.e., the host T-cell proliferative responses (Fig. 1b) . Although not significant, the proportion of BrdU+ cells within the rosette at 18 and 24 h tended to be higher in the test rats than in the control rats (Fig. 1f) .



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  		Figure 6. Formation of clusters by host DCs in the T-cell area of hepatic LNs at 3 days postinjection, determined by triple immunostaining for macrophages and lymphocytes (brown), host MHC II antigens (blue), and BrdU (red). Note that host DC (blue, arrows) are surrounded by one or two BrdU+ host cells (red nuclei, arrows), forming many rosettes. The phenotype of the BrdU+ cells was not determined because of weak brown staining. Magnification, x500._art>

 
Fate of donor DCs after cardiac allotransplantation
Donor DCs could be readily identified as donor-type MHC II-single-positive DCs lacking macrophage and lymphocyte markers. A small but significant number of donor DCs were found in the T-cell areas of host spleens (PALS) at day 2 (Fig. 7a ) and of hepatic LNs at days 2 and 3 of transplantation, indicating their blood-borne migration [7 ]. In addition, in the PALS of a day 2 spleen, donor MHC II+ dotty fragments were detected within the host MHC II+ cells with dendritic morphology (Fig. 8a andb), in the same fashion as the DC transfer study. These cells were also considered to be mostly host IDCs because of their morphology and location.



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  		Figure 7. Kinetics of donor DCs, host proliferative response, and rosette formation in the host spleen after cardiac allotransplantation. Values are means ± SD (n = 3). (a) Absolute number of migrated donor DCs, identified as donor MHC II-single-positive cells in the PALS of the 8µm-thick mid-cross sections. (b) Proliferative response in the PALS, expressed as the number of BrdU+ cells per square millimeter of 6µm-thick sections. (c) Absolute number of the donor rosettes containing either one BrdU+ cell or two or more BrdU+ cells in the PALS of 8µm-thick mid-cross sections. (d) Number of host rosettes containing either one BrdU+ cells or two or more BrdU+ cells per square millimeter of 4µm-thick sections. (e) Ratio of number of BrdU+ cells within the rosette to the total number of BrdU+ cells.

 


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  		Figure 8. Migration of donor DCs to the T-cell area of the host spleen (PALS) at 2 days after cardiac allotransplantation. Double immunostaining for donor MHC II (brown) and host MHC II (blue) was performed. Donor MHC II dotty fragments (arrowheads) are within host MHC II+ cells with dendritic morphology in panel a (magnification, x250)and, at a higher magnification (x500), in panel b, indicating phagocytosis of donor DC by host putative IDCs. Two donor DCs can be also seen (arrow) in panel a. (c) Sections of the same group, triple immunostained for donor MHC II (blue), BrdU (red), and type IV collagen (brown). One donor DC (blue) is surrounded by three host BrdU+ cells, forming a typical rosette (arrow).

 
Rosette formation after cardiac allotransplantation
A T-cell proliferative response was readily observed in the spleen, with a peak at day 3 (Fig. 7b) . Donor DCs formed rosettes in splenic PALS only at day 2 (Fig. 7c and 8c) . Host MHC II+ cells without macrophage and B-cell markers in the PALS formed clear rosettes (Fig. 9a 9b ). These cells were also negative for T-cell markers (data not shown). The number of rosettes with two or more BrdU+ cells as well as the number with one BrdU+ cell increased significantly from day 2 after transplantation, reached its peak at day 3 or 4, and then quickly decreased by day 5 (Fig. 7d) . More than half of the BrdU+ cells were inside the rosettes from days 2 to 4 (Fig. 7e) . These results demonstrate that the kinetics of the T-cell proliferation response within the PALS is confined to the rosettes of host putative IDCs.



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  		Figure 9. Formation of rosettes by host DCs (blue) and host BrdU+ cells in the PALS. Triple immunostaining for macrophages and B cells (brown), host MHC II antigens (blue), and BrdU (red) was performed. (a) A whole view of white pulp in which PALS (P) can be readily discriminated as a non-B-cell area. A; central artery; F, B-cell follicle. Magnification, x125. (b) A high-power view of typical host rosettes (arrows) surrounded by many host proliferating cells (red). Magnification, x500.

 
In the grafted heart tissues, formation of rosettes by either predominantly donor (Fig. 10 ) or host MHC II-single-positive cells and host proliferating cells was observed from days 2 to 4. In the regraft study, rosette formation by the remobilized first-host DCs and second-host BrdU+ cells was readily observed in the spleen (Fig. 11 ) and hepatic LNs.



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  		Figure 10. Formation of rosettes by donor MHC II single-positive cells and host proliferating cells in graft tissues at day 2 after cardiac transplantation. Triple immunostaining for macrophages and B cells (brown), donor MHC II antigen (blue), and BrdU (red) was performed. Note that two donor DCs are surrounded by BrdU+ cells. Magnification, x500.

 


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  		Figure 11. Formation of rosettes by remobilized first-host DCs with second-host BrdU+ cells in splenic PALS 38 h after the regraft operation. Triple immunostaining for macrophages and lymphocytes (brown), first-host MHC II (blue), and BrdU (red) was performed. The phenotype of BrdU cells was not determined because of weak brown staining. Magnification, x500.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, the in situ allogeneic immune response in host lymphoid tissues was visualized by using rat DC transfer and cardiac transplantation models. We revealed that the donor DCs formed cell clusters (rosettes) with proliferating host T cells at the DC migration sites, i.e., the T-cell areas of the host hepatic LNs (paracortex) and spleen (PALS). In and around these rosettes, the T-cell proliferative response started. Concomitantly, we found a rapid disintegration of donor DCs and observed donor MHC II+ fragments within the cytoplasm of host MHC II+ cells. Furthermore, host-resident DCs (IDCs) formed clusters with proliferating T cells in a more expanded, diffuse fashion in the T-cell area. The kinetics of host cluster formation correlated well with that of the T-cell proliferative response in these tissues.

What is the significance of the donor rosette in the allosensitization process? Initiation of the T-cell proliferative response within the donor rosette demonstrates that T-cell activation takes place within this structure. Furthermore, the clear expression of costimulatory molecules by donor DCs and the electron-microscopic observation of intimate contact of donor DCs with host lymphoblasts are indicative of the occurrence of significant cellular interactions. Therefore, the results of this study have revealed that the donor rosette is a structure in which antigen-specific T cells are triggered and start clonal expansion, resulting in direct allosensitization. Rosette formation by DCs for allosensitization is a novel finding. Formation of rosettes by donor MHC II+ cells and host proliferating cells in the host spleen in a rat liver allograft model was reported [19 ], but these MHC II+ cells were not proven to be DCs. Rosette formation by splenic IDCs during the immune response to a superantigen [20 ] is the only relevant report, to the best of our knowledge.

At the late stage, the sensitization process may be followed by the killing process of DCs as a target of sensitized T cells [21 , 22 ]. In this respect, at 48 h after DC injection, the formation of rosettes by the remaining donor DCs was even exaggerated in areas within which donor DCs were degenerating. Since very few DCs exit from the LNs [6 , 11 ], these findings suggest that proliferating sensitized T cells quickly kill and eliminate donor DCs. On the other hand, the cluster of donor DCs with BrdU- host T cells evident during the early stages of sensitization is probably, for the most part, a site for selection of antigen-specific T cells by DCs through nonspecific binding [21 , 23 ].

Host DCs may play a crucial role in the allogeneic response. Phagocytosis of donor MHC II fragments by host MHC II+ cells, which are mostly host IDCs, is a novel finding. The IDCs in lymphoid tissues correspond to mature DCs in vitro, which should have little phagocytic activity [23 ]. However, in vivo, these cells bear the phagocytosis-related ED1 antigen in rats [17 ] and are CD68+ in mice [24 ], and they were reported to exhibit phagocytic activity in rats [25 ]. Therefore, together with the host rosette formation, the results indicate that host IDCs quickly phagocytose, and presumably process and present, donor MHC antigens to host T cells in an indirect manner. In this respect, when live allogeneic DCs, but not other cell types undergoing apoptosis, were intradermally injected into mice, a majority of host IDCs in the draining LNs expressed complexes formed between host MHC II and a donor MHC II peptide [4 ]. This indicates that among leukocytes, live allogeneic DCs are phagocytosed and most efficiently processed and presented by host DCs, thus supporting our interpretation. Since even a killing activity of tumor cells by rat splenic DCs has been recently reported [26 , 27 ], IDCs might also directly kill the donor DCs, which might eventually facilitate phagocytosis. Alternatively, host DCs might also simply support T-cell growth in the rosettes as nurse cells providing an immunoproliferative microenvironment [28 , 29 ]. This may explain the widespread proliferation, because sensitized T cells may move around the T-cell area and cluster with nursing host DCs at remote sites where these cells could continue to proliferate.

Concerning the actual sensitization process in the rat cardiac transplantation model, we have found that intrahost sensitization is the major pathway [7 ]. Host T cells may be sensitized via the direct pathway by migrated donor DCs and/or via the indirect pathway by host IDCs in the T-cell area DCs, which ingested migrated donor cells as described above. Additionally, the host DCs, which had been recruited to the graft tissues and then were remobilized to the host spleen and hepatic LNs, might also be responsible for the indirect pathway, as shown by their rosette formation. Alternatively, graft-derived soluble MHC antigens may also play a role in the indirect pathway [30 ]. This is rather unlikely, at least in the case of the hepatic LNs, because only antigens in a cell-laden form, not free antigens, can selectively accumulate in the particular LNs. Intragraft sensitization may also occur via both donor DCs (direct) and recruited host DCs (indirect) in the graft tissues, as revealed by their rosette formation. Formation of clusters by donor MHC II+ cells and lymphocytes in the graft was also reported [31 ]. Together, these results suggest that in rat heart transplantation the host rats are allosensitized mainly in the host spleen and hepatic LN either by migrated graft-derived DCs (direct intrahost sensitization), by host IDCs or by remobilized host DCs (indirect intrahost sensitization). Intragraft sensitization by either donor DCs or recruited host DCs within the graft may also contribute in part.

In conclusion, donor DCs and, presumably, host IDCs sensitize host T cells within rosettes in an direct and indirect manner, respectively. At the late stage of sensitization, the donor rosettes become sites for killing of donor DCs as target cells. Migrated donor DCs are readily phagocytosed and may be most efficiently processed and presented by host IDCs. The host DCs may also simply support T-cell growth as nurse cells in the rosette microenvironment. Rosette formation can be used as a useful parameter for detecting a site of antigen presentation in various pathological states involving immune responses.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr. Hiromitsu Kimura for teaching us a heart grafting technique and for discussions and to Prof. Shigeo Ekino for his encouragement and support. This work was supported by a Grant-in-Aid for Scientific Research (B) No. 12470004 from the Japanese Ministry of Education, Science, and Culture.

Received September 22, 2000; revised December 19, 2000; accepted December 20, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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