Hisashi Ueta

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

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 Peyers 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.)

Trafficking of recirculating lymphocytes in the rat liver: rapid transmigration into the portal area and then to the hepatic lymph

Background: We have investigated how recirculating lymphocytes patrol the liver in a normal steady state.

Predominant donor CD103+CD8+ T cell infiltration into the gut epithelium during acute GvHD: a role of gut lymph nodes

The existence of donor effector cell subsets responsible for either gut or skin graft-versus-host disease (GvHD) is still undetermined. We examined the trafficking and role of donor CD8(+) intra-epithelial lymphocytes (IELs) in the gut and skin epithelia concerning alpha E beta 7 integrin (CD103) expression, using a rat acute lethal GvHD model. Most CD103(+) donor cells were CD8(+) and showed a proliferative activity in the target epithelia. On the other hand, activated donor T cells in the host lymphoid tissues did not express CD103, indicating the presence of CD8(+) IEL precursors in the lymphoid tissues that may up-regulate CD103 only after migrating to the target organs. At the late stage of GvHD, while >80% of the donor CD8(+) IELs were CD103(+) in the gut epithelium, both CD4(+) and CD103(+)CD8(+) T cells evenly accumulated in the skin epidermis. The CD103 expression by donor CD8(+) IELs especially in the gut was also correlated with the clinical GvHD manifestations. Furthermore, the selective removal of gut lymph nodes (LNs) but not skin LNs suppressed the infiltration of CD103(+) donor IELs in the gut and alleviated intestinal GvHD. In conclusion, CD103(+)CD8(+) donor T cells predominantly infiltrate into the gut epithelium and are responsible for the manifestations of intestinal GvHD. This pathology is at least partly dependent on the gut LNs.

Two immunogenic passenger dendritic cell subsets in the rat liver have distinct trafficking patterns and radiosensitivities

The aim of this study was to investigate the trafficking patterns, radiation sensitivities, and functions of conventional dendritic cell (DC) subsets in the rat liver in an allotransplantation setting. We examined DCs in the liver, hepatic lymph, and graft tissues and recipient secondary lymphoid organs after liver transplantation from rats treated or untreated by sublethal irradiation. We identified two distinct immunogenic DC subsets. One was a previously reported population that underwent blood‐borne migration to the recipients secondary lymphoid organs, inducing systemic CD8+ T‐cell responses; these DCs are a radiosensitive class II major histocompatibility complex (MHCII)+CD103+CD172a+CD11b−CD86+ subset. Another was a relatively radioresistant MHCII+CD103+CD172a+CD11b+CD86+ subset that steadily appeared in the hepatic lymph. After transplantation, the second subset migrated to the parathymic lymph nodes (LNs), regional peritoneal cavity nodes, or persisted in the graft. Irradiation completely eliminated the migration and immunogenicity of the first subset, but only partly suppressed the migration of the second subset and the CD8+ T‐cell response in the parathymic LNs. The grafts were acutely rejected, and intragraft CD8+ T‐cell and FoxP3+ regulatory T‐cell responses were unchanged. The radioresistant second subset up‐regulated CD25 and had high allostimulating activity in the mixed leukocyte reaction, suggesting that this subset induced CD8+ T‐cell responses in the parathymic LNs and in the graft by the direct allorecognition pathway, leading to the rejection. Conclusion: Conventional rat liver DCs contain at least two distinct immunogenic passenger subsets: a radiosensitive blood‐borne migrant and a relatively radioresistant lymph‐borne migrant. LNs draining the peritoneal cavity should be recognized as a major site of the intrahost T‐cell response by the lymph‐borne migrant. This study provides key insights into liver graft rejection and highlights the clinical implications of immunogenic DC subsets. (HEPATOLOGY 2012)

Generation of anti-human DEC205/CD205 monoclonal antibodies that recognize epitopes conserved in different mammals

DEC205/CD205 is a C-type multilectin receptor, expressed highly in dendritic cells (DCs). Previous efforts to generate anti-human DEC205 (anti-hDEC205) monoclonal antibodies (mAbs) from mice immunized with subdomain proteins of hDEC205 resulted in a few mAbs. Recently, we expressed and utilized a full-length extracellular domain protein of hDEC205 to successfully generate 5 strong anti-hDEC205 mAbs from mice. In this study, DEC205 knockout (KO) mice were immunized with this full-length extracellular domain protein of hDEC205. One of the 3 immunized DEC205 KO mice was chosen for the highest anti-hDEC205 titer by flow cytometric analysis of serum samples on CHO cells stably expressing hDEC205 (CHO/hDEC205 cells) and used for hybridoma fusion. From a single fusion, more than 400 anti-hDEC205 hybridomas were identified by flow cytometric screen with CHO/hDEC205 cells, and a total of 115 hybridomas secreting strong anti-hDEC205 mAb were saved and named HD1 through HD115. To characterize in detail, 10 HD mAbs were chosen for superior anti-hDEC205 reactivity and further subjected to cloning and purification. Interestingly, out of those 10 chosen anti-hDEC205 HD mAbs, 5 mAbs were also strongly reactive to mouse DEC205 while 8 mAbs were found to stain DEC205(+) DCs on monkey spleen sections. In addition, we also identified that HD83, one of the 10 chosen HD mAbs, stains DEC205(+) DCs in rat spleen and lymph node. Therefore, by immunizing DEC205 KO mice with a full-length extracellular domain protein of hDEC205, we generated a large number of strong anti-hDEC205 mAbs many of which are cross-species reactive and able to visualize DEC205(+) DCs in lymphoid tissues of other mammals.

A novel multicolor immunostaining method using ethynyl deoxyuridine for analysis of in situ immunoproliferative response

Abstract Immune responses are generally accompanied by antigen presentation and proliferation and differentiation of antigen-specific lymphocytes (immunoproliferation), but analysis of these events in situ on tissue sections is very difficult. We have developed a new method of simultaneous multicolor immunofluorescence staining for immunohistology and flow cytometry using a thymidine analogue, 5-ethynyl-2′-deoxyuridine (EdU). Because of the small size of azide dye using click chemistry and elimination of DNA denaturation steps, EdU staining allowed for immunofluorescence staining of at least four colors including two different markers on a single-cell surface, which is impossible with the standard 5-bromo-2′-deoxyuridine method. By using two rat models, successfully detected parameters were the cluster of differentiation antigens including phenotypic and functional markers of various immune cells, histocompatibility complex antigens, and even some nuclear transcription factors. Proliferating cells could be further sorted and used for RT-PCR analysis. This method thus enables functional in situ time-kinetic analysis of immunoproliferative responses in a distinct domain of the lymphoid organs, which are quantitatively confirmed by flow cytometry.

Role of Uterine Contraction in Regeneration of the Murine Postpartum Endometrium

ABSTRACT The endometrium undergoes continuous repair and regeneration without scarring throughout the reproductive life of women. However, the mechanisms responsible for this complete restoration remain mostly unexplored. We hypothesized that the ischemic state and local hypoxia present after parturition may create a special microenvironment for endometrial healing, and that this ischemia might be caused by reduction in organ volume via postpartum uterine contraction. Here, we developed a mouse model using a combination of cesarean section and the administration of a beta 2 adrenergic receptor agonist (ritodrine hydrochloride) in postpartum mice that had been ovariectomized to exclude the effect of ovarian hormones. Our results revealed that transient hypoxia indeed occurred in postpartum uteri. Furthermore, we found that the number of M2 macrophages, which play a central role in wound healing, peaked on Postpartum Day 3 and gradually decreased thereafter in hypoxic injury sites. Almost concurrently, significant upregulation of vascular endothelial growth factor and transforming growth factor beta (TGFbeta) was observed. In particular, the antifibrotic factor TGFbeta3 was released during the endometrial healing process. These changes were significantly suppressed by inhibition of uterine contraction. Taken together, these results suggest that uterine contraction is essential, not only for hemostasis, but also for endometrial regeneration, leading to a process that involves the activation of macrophages, increased endometrial cell proliferation, and upregulation of nonfibrotic growth factors. This study paves the way to a novel approach for investigating the process of scarless wound healing.

Direct evidence for activated CD8+ T cell transmigration across portal vein endothelial cells in liver graft rejection

BackgroundLymphocyte recruitment into the portal tract is crucial not only for homeostatic immune surveillance but also for many liver diseases. However, the exact route of entry for lymphocytes into portal tract is still obscure. We investigated this question using a rat hepatic allograft rejection model.MethodsA migration route was analyzed by immunohistological methods including a recently developed scanning electron microscopy method. Transmigration-associated molecules such as selectins, integrins, and chemokines and their receptors expressed by hepatic vessels and recruited T-cells were analyzed by immunohistochemistry and flow cytometry.ResultsThe immunoelectron microscopic analysis clearly showed CD8β+ cells passing through the portal vein (PV) endothelia. Furthermore, the migrating pathway seemed to pass through the endothelial cell body. Local vascular cell adhesion molecule-1 (VCAM-1) expression was induced in PV endothelial cells from day 2 after liver transplantation. Although intercellular adhesion molecule-1 (ICAM-1) expression was also upregulated, it was restricted to sinusoidal endothelia. Recipient T-cells in the graft perfusate were CD25+CD44+ICAM-1+CXCR3+CCR5– and upregulated α4β1 or αLβ2 integrins. Immunohistochemistry showed the expression of CXCL10 in donor MHCIIhigh cells in the portal tract as well as endothelial walls of PV.ConclusionsWe show for the first time direct evidence of T-cell transmigration across PV endothelial cells during hepatic allograft rejection. Interactions between VCAM-1 on endothelia and α4β1 integrin on recipient effector T-cells putatively play critical roles in adhesion and transmigration through endothelia. A chemokine axis of CXCL10 and CXCR3 also may be involved.

Graft‐Versus‐Host Disease Following Liver Transplantation: Development of a High‐Incidence Rat Model and a Selective Prevention Method

Graft‐versus‐host disease (GvHD) following liver transplantation (LT) is a rare but serious complication with no presently available animal model and no preventive measures. To develop a rat model of GvHD after LT (LT‐GvHD), we preconditioned hosts with sublethal irradiation plus reduction of natural killer (NK) cells with anti‐CD8α mAb treatment, which invariably resulted in acute LT‐GvHD. Compared with those in the peripheral counterpart, graft CD4+CD25− passenger T cells showed lower alloreactivities in mixed leukocyte culture. Immunohistology revealed that donor CD4+ T cells migrated and formed clusters with host dendritic cells in secondary lymphoid organs, with early expansion and subsequent accumulation in target organs. For selectively preventing GvHD, donor livers were perfused ex vivo with organ preservation media containing anti‐TCRαβ mAb. T cell–depleted livers almost completely suppressed clinical GvHD such that host rats survived for >100 days. Our results showed that passenger T cells could develop typical LT‐GvHD if resistant cells such as host radiosensitive cells and host radioresistant NK cells were suppressed. Selective ex vivo T cell depletion prevented LT‐GvHD without affecting host immunity or graft function. This method might be applicable to clinical LT in prediagnosed high‐risk donor–recipient combinations and for analyzing immunoregulatory mechanisms of the liver.

Visualizing the Rapid and Dynamic Elimination of Allogeneic T Cells in Secondary Lymphoid Organs

Allogeneic organ transplants are rejected by the recipient immune system within several days or weeks. However, the rejection process of allogeneic T (allo-T) cells is poorly understood. In this study, using fluorescence-based monitoring and two-photon live imaging in mouse adoptive transfer system, we visualized the fate of allo-T cells in the in vivo environment and showed rapid elimination in secondary lymphoid organs (SLOs). Although i.v. transferred allo-T cells efficiently entered host SLOs, including lymph nodes and the spleen, ∼70% of the cells had disappeared within 24 h. At early time points, allo-T cells robustly migrated in the T cell area, whereas after 8 h, the numbers of arrested cells and cell fragments were dramatically elevated. Apoptotic breakdown of allo-T cells released a large amount of cell debris, which was efficiently phagocytosed and cleared by CD8+ dendritic cells. Rapid elimination of allo-T cells was also observed in nu/nu recipients. Depletion of NK cells abrogated allo-T cell reduction only in a specific combination of donor and recipient genetic backgrounds. In addition, F1 hybrid transfer experiments showed that allo-T cell killing was independent of the missing-self signature typically recognized by NK cells. These suggest the presence of a unique and previously uncharacterized modality of allorecognition by the host immune system. Taken together, our findings reveal an extremely efficient and dynamic process of allogeneic lymphocyte elimination in SLOs, which could not be recapitulated in vitro and is distinct from the rejection of solid organ and bone marrow transplants.