Suppression of liver transplant rejection by anti-donor MHC antibodies via depletion of donor immunogenic dendritic cells.

BACKGROUND: We previously found two distinct passenger dendritic cell (DC) subsets in the rat liver that played a central role in the liver transplant rejection. In addition, a tolerance-inducing protocol, donor-specific transfusion (DST), triggered systemic polytopical production of depleting alloantibodies to donor class I MHC (MHCI) antigen (DST-antibodies). METHODS: We examined the role of DST-antibodies in the trafficking of graft DC subsets and the alloresponses in a rat model. We also examined an anti-donor class II MHC (MHCII) antibody that recognizes donor DCs more selectively. RESULTS: Preoperative transfer of DST-antibodies or DST pretreatment eliminated all passenger leukocytes, including both DC subsets and depleted the sessile DCs in the graft to ~20% of control. The CD172a+CD11b/c+ immunogenic subset was almost abolished. The intrahost direct or semi-direct allorecognition pathway was successfully blocked, leading to a significant suppression of the CD8+ T-cell response in the recipient lymphoid organs and the graft with delayed graft rejection. Anti-donor MHCII antibody had similar effects without temporary graft damage. Although DST pretreatment had a priming effect on the proliferative response of recipient regulatory T cells, DST-primed sera and the anti-donor MHCII antibody did not. CONCLUSION: DST-antibodies and anti-donor MHCII antibodies could suppress the CD8+ T-cell-mediated liver transplant rejection by depleting donor immunogenic DCs, blocking the direct or semi-direct pathways of allorecognition. Donor MHCII-specific antibodies may be applicable as a selective suppressant of anti-donor immunity for clinical liver transplantation without the cellular damage of donor MHCII- graft cells and recipient cells.

Selective involution of thymic medulla by cyclosporine A with a decrease of mature thymic epithelia, XCR1+ dendritic cells, and epithelium-free areas containing Foxp3+ thymic regulatory T cells.

Immunosuppressive drugs such as cyclosporine A (CSA) can disrupt thymic structure and functions, ultimately inducing syngeneic/autologous graft-versus-host disease together with involuted medullas. To elucidate the effects of CSA on the thymus more precisely, we analyzed the effects of CSA on the thymus and T cell system using rats. In addition to confirming the phenomena already reported, we newly found that the proportion of recent thymic emigrants also greatly decreased, suggesting impaired supply. Immunohistologically, the medullary thymic epithelial cells (mTECs) presented with a relative decrease in the subset with a competent phenotype and downregulation of class II major histocompatibility complex molecules. In control rats, thymic dendritic cells (DCs) comprised two subsets, XCR1+SIRP1α-CD4- and XCR1-SIRP1α+CD4+. The former had a tendency to selectively localize in the previously-reported epithelium-containing areas of the rat medullas, and the number was significantly reduced by CSA treatment. The epithelium-free areas, another unique domains in the rat medullas, contained significantly more Foxp3+ thymic Tregs. With CSA treatment, the epithelium-free areas presented strong involution, and the number and distribution of Tregs in the medulla were greatly reduced. These results suggest that CSA inhibits the production of single-positive thymocytes, including Tregs, and disturbs the microenvironment of the thymic medulla, with a decrease of the competent mTECs and disorganization of epithelium-free areas and DC subsets, leading to a generation of autoreactive T cells with selective medullary involution.

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.

Single blood transfusion induces the production of donor-specific alloantibodies and regulatory T cells mainly in the spleen.

Donor-specific blood transfusion is known to induce alloresponses and lead to immunosuppression. We examined their underlying mechanisms by employing fully allogeneic rat combinations. Transfused recipients efficiently produced alloantibodies of the IgM and IgG subclasses directed against donor class I MHC. The recipients exhibited active expansion of CD4+ T cells and CD4+FOXP3+ regulatory T cells (Treg cells), followed by CD45R+ B cells and IgM+ or IgG subclass+ antibody-forming cells mainly in the spleen. From 1.5 days, the resident MHCII+CD103+ dendritic cells (DCs) in the splenic T-cell area, periarterial lymphocyte sheath, formed clusters with recipient BrdU+ or 5-ethynyl-2'-deoxyuridine+ cells, from which the proliferative response of CD4+ T cells originated peaking at 3-4 days. Transfusion-induced antibodies had donor passenger cell-depleting activity in vitro and in vivo and could suppress acute GvH disease caused by donor T cells. Furthermore, Treg cells significantly suppressed mixed leukocyte reactions in a donor-specific manner. In conclusion, single blood transfusion efficiently induced a helper T-cell-dependent anti-donor class I MHC antibody-forming cell response with immunoglobulin class switching, and a donor-specific Treg cell response mainly in the spleen, probably by way of the indirect allorecognition via resident DCs. These antibodies and Treg cells may be involved, at least partly, in the donor-specific transfusion-induced suppression of allograft rejection.

Rapid immunosurveillance by recirculating lymphocytes in the rat intestine: critical role of unsulfated sialyl-Lewis X on high endothelial venules of the Peyer's patches.

Naive lymphocytes systemically recirculate for immunosurveillance inspecting foreign antigens and pathogens in the body. Trafficking behavior such as the migration pathway and transit time within the gastrointestinal tract, however, remains to be elucidated. Rat thoracic duct lymphocytes (TDLs) were transferred to a congeneic host that had undergone mesenteric lymphadenectomy. The migration pathway was investigated using newly developed four-color immunohistochemistry and immunofluorescence. Donor TDLs showed rapid transition in gut tissues from which they emerged in mesenteric lymph around 4 h after intravenous injection. Immunohistochemistry showed that donor TDLs predominantly transmigrated across high endothelial venules (HEVs) at the interfollicular area of the Peyer's patches (PPs), then exited into the LYVE-1+ efferent lymphatics, that were close to the venules. The rapid recirculation depended largely on the local expression of unsulfated sialyl-Lewis X on these venules where putative dendritic cells (DCs) were associated underneath. Recruited naive T cells briefly made contact with resident DCs before exiting to the lymphatics in the steady state. In some transplant settings, however, the T cells retained contact with DCs and were sensitized and differentiated into activated T cells. In conclusion, we directly demonstrated that lymphocyte recirculation within the gut is a very rapid process. The interfollicular area of PPs functions as a strategically central site for rapid immunosurveillance where HEVs, efferent lymphatics and resident DCs converge. PPs can, however, generate alloreactive T cells, leading to exacerbation of graft-versus-host disease or gut allograft rejection.

Anatomical basis for simultaneous block of greater and third occipital nerves, with an ultrasound-guided technique.

PURPOSE: In some headache disorders, for which the greater occipital nerve block is partly effective, the third occipital nerve is also suggested to be involved. We aimed to establish a simple technique for simultaneously blocking the greater and third occipital nerves. METHODS: We performed a detailed examination of dorsal neck anatomy in 33 formalin-fixed cadavers, and deduced two candidate target points for blocking both the greater and third occipital nerves. These target points were tested on three Thiel-fixed cadavers. We performed ultrasound-guided dye injections into these points, examined the results by dissection, and selected the most suitable injection point. Finally, this target point was tested in three healthy volunteers. We injected 4 ml of local anesthetic and 1 ml of radiopaque material at the selected point, guided with a standard ultrasound system. Then, the pattern of local anesthetic distribution was imaged with computed tomography. RESULTS: We deduced that the most suitable injection point was the medial head of the semispinalis capitis muscle at the C1 level of the cervical vertebra. Both nerves entered this muscle, in close proximity, with little individual variation. In healthy volunteers, an anesthetic injected was confined to the muscle and induced anesthesia in the skin areas innervated by both nerves. CONCLUSIONS: The medial head of the semispinalis capitis muscle is a suitable landmark for blocking the greater and third occipital nerves simultaneously, by which occipital nerve involvement in various headache disorders may be rapidly examined and treated.

Expression of area-specific M2-macrophage phenotype by recruited rat monocytes in duct-ligation pancreatitis.

Acute pancreatitis remains a disease of uncertain pathogenesis and no established specific therapy. Previously, we found a predominant increase and active proliferation of macrophages in the inflamed tissues of a rat duct-ligation pancreatitis model. To analyze the origin and possible role of these macrophages, we investigated their in situ cellular kinetics in a rat model of duct-ligation pancreatitis using a recently established method of multicolor immunostaining for macrophage markers and for proliferating cells with ethynyl deoxyuridine. To detect monocyte-derived macrophages, green fluorescent protein-transgenic (GFP(+)) leukocytes were transferred to monocyte-depleted recipients. In the inflamed pancreas, infiltrating macrophages were mainly two phenotypes, CD68(+)CD163(-) round cells and CD68(+)CD163(+) large polygonal cells, both of which showed active proliferation. In the interlobular area, the proportions of CD68(+)CD163(low) and CD68(+)CD163(high) cells increased over time. Most expressed the M2-macrophage markers CD206 and arginase 1. In contrast, in the interacinar area, CD68(+) cells did not upregulate CD163 and CD206, but ~30 % of them expressed the M1 marker nitric oxide synthase 2 on day 4. GFP(+)-recruited cells were primarily CD68(+)CD163(-) monocytes on day 1 and showed phenotypic changes similar to those of the monocyte non-depleted groups. In conclusion, infiltrating macrophages mostly formed two distinct subpopulations in different areas: monocyte-derived macrophages with the M2 phenotype in the interlobular area or non-M2 phenotype in the interacinar area. Involvement of resident macrophages might be minor in this model. These results are the first demonstration of an upregulated M2 phenotype in rat inflammatory monocytes, which may promote tissue repair.

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

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.

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.

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

UNLABELLED: 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 recipient's 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.

Temporal and spatial dynamics of immune cells in spontaneous liver transplant tolerance.

The liver has long been deemed a tolerogenic organ. We employed high-dimensional mass cytometry and immunohistochemistry to depict the temporal and spatial dynamics of immune cells in the spleen and liver in a murine model of spontaneous liver allograft acceptance. We depicted the immune landscape of spontaneous liver tolerance throughout the rejection and acceptance stages after liver transplantation and highlighted several points of importance. Of note, the CD4+/CD8+ T cell ratio remained low, even in the tolerance phase. Furthermore, a PhenoGraph clustering analysis revealed that exhausted CD8+ T cells were the most dominant metacluster in graft-infiltrating lymphocytes (GILs), which highly expressed the costimulatory molecule CD86. The temporal and spatial dynamics of immune cells revealed by high-dimensional analyses enable a fine-grained analysis of GIL subsets, contribute to new insights for the discovery of immunological mechanisms of liver tolerance, and provide potential ways to achieve clinical operational tolerance after liver transplantation.

Utility of nano-sized, water-in-oil emulsion as a sustained release formulation of glycyrrhizin.

The aim of this study was to determine the efficiency of nano-sized water-in-oil (w/o) emulsions that encapsulate glycyrrhizin (GZ) (Rp-I) as a sustained release formulation for subcutaneous administration. Four formulations were assessed in rats for 8-72 h: nano-sized water-in-oil (w/o) emulsion encapsulating GZ (Rp-I), GZ aqueous solution (Rp-II), oil-in-water (o/w) emulsion containing GZ (Rp-III), and w/o emulsion containing solid GZ (Rp-IV). All had a GZ concentration of 150 mg/ml. Over an 8-h period, GZ elimination in bile after subcutaneous administration of Rp-I, Rp-II, Rp-III, and Rp-IV (50 mg/kg GZ) was 10.8%, 97.0%, 81.0%, and 7.1%, respectively. The elimination of GZ into bile after the administration of Rp-IV was the lowest (30.5%) at the 72-h endpoint, dropping significantly from 48 to 72 h. On the other hand, the elimination rate of GZ after the administration of Rp-I was sustained at a constant level (1.8-2.1 mg/24 h) over 72 h. GZ concentration in liver at 72 h in Rp-I was highest (19.9 µg/g tissue) among the four formulations, suggesting that the release of GZ from the Rp-I formulation is constant, at least up to 72 h after administration. These results suggest that a nano-sized w/o emulsion is useful as a sustained release formulation for long-term therapy of chronic hepatitis.

Extracellular ATP Limits Homeostatic T Cell Migration Within Lymph Nodes.

Whereas adenosine 5'-triphosphate (ATP) is the major energy source in cells, extracellular ATP (eATP) released from activated/damaged cells is widely thought to represent a potent damage-associated molecular pattern that promotes inflammatory responses. Here, we provide suggestive evidence that eATP is constitutively produced in the uninflamed lymph node (LN) paracortex by naïve T cells responding to C-C chemokine receptor type 7 (CCR7) ligand chemokines. Consistently, eATP was markedly reduced in naïve T cell-depleted LNs, including those of nude mice, CCR7-deficient mice, and mice subjected to the interruption of the afferent lymphatics in local LNs. Stimulation with a CCR7 ligand chemokine, CCL19, induced ATP release from LN cells, which inhibited CCR7-dependent lymphocyte migration in vitro by a mechanism dependent on the purinoreceptor P2X7 (P2X7R), and P2X7R inhibition enhanced T cell retention in LNs in vivo. These results collectively indicate that paracortical eATP is produced by naïve T cells in response to constitutively expressed chemokines, and that eATP negatively regulates CCR7-mediated lymphocyte migration within LNs via a specific subtype of ATP receptor, demonstrating its fine-tuning role in homeostatic cell migration within LNs.

The microstructure of secondary lymphoid organs that support immune cell trafficking.

Immune cell trafficking in the secondary lymphoid organs is crucial for an effective immune response. Recirculating T cells constantly patrol not only secondary lymphoid organs but also the whole peripheral organs. Thoracic duct lymphocytes represent an ideal cell source for analyzing T cell trafficking: high endothelial venules (HEVs) allow recirculating lymphocytes to transmigrate from the blood directly, and recirculating T cells form a cluster with dendritic cells (DCs) to survey antigen invasions even in a steady state. This cluster becomes an actual site for the antigen presentation when DCs have captured antigens. On activation, effector and memory T cells differentiate into several subsets that have different trafficking molecules and patterns. DCs also migrate actively in a manner depending upon their maturational stages. Danger signals induce the recruitment of several DC precursor subsets with different trafficking patterns and functions. In this review, we describe general and specialized structures of the secondary lymphoid organs for the trafficking of T cells and DCs by a multicolor immunoenzyme staining technique. The lymph nodes, spleen, and Peyer's patches of rats were selected as the major representatives. In vivo trafficking of subsets of T cells and DCs within these organs under steady or emergency states are shown and discussed, and unsolved questions and future prospects are also considered.

Monotherapy With Anti-CD70 Antibody Causes Long-Term Mouse Cardiac Allograft Acceptance With Induction of Tolerogenic Dendritic Cells.

Allograft rejection has been an obstacle for the long-term survival of patients. CD70, a tumor necrosis factor (TNF) family member critically expressed on antigen-presenting cells and strongly but transiently up-regulated during lymphocyte activation, represents an important co-stimulatory molecule that induces effective T cell responses. We used a mouse heterotopic cardiac transplantation model to evaluate the effects of monotherapy with the antibody targeting mouse CD70 (FR70) on transplantation tolerance and its immunoregulatory activity. FR70-treated C3H recipient mice permanently accepted B6 fully mismatched cardiac allografts. Consistent with the graft survival, the infiltration of CD8+ T cells in the graft was reduced, dendritic cells were differentiated into a tolerogenic status, and the number of regulatory T cells was elevated both in the graft and the recipient's spleen. In addition, naïve C3H given an adoptive transfer of spleen cells from the primary recipients with FR70 treatment accepted a heart graft from a matching B6 donor but not third-party BALB/c mice.　Our findings show that treatment with FR70 induced regulatory cells and inhibited cytotoxic T cell proliferation, which led to long-term acceptance of mouse cardiac allografts. These findings highlight the potential role of anti-CD70 antibodies as a clinically effective treatment for allograft rejection.

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

UNLABELLED: 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.

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.

Simultaneous immunohistochemical detection of gangliosides and neuronal markers in paraformaldehyde-fixed nervous tissues by acetone etching.

A need for identifying ganglioside-positive cells with neuronal markers prompted us to establish a reliable method for double or triple immunostaining nervous tissues. Perfusion fixation with paraformaldehyde is typically performed for the routine immunostaining of various neuronal markers but is not suitable for immunostaining gangliosides. Acetone fixation of fresh cryosections is frequently used for ganglioside immunodetection; thus, we tested the effect of acetone treatment for unmasking the antigen epitope of gangliosides (acetone etching) on sections of paraformaldehyde-fixed nervous tissue from rats. Acetone etching significantly retrieved ganglioside immunoreactivity while preserving the immunoreactivity of neuronal markers. Various combinations of gangliosides and neuronal markers could be double-stained by the immunoenzyme method or triple-stained by the immunofluorescence method. This new method may provide additional information regarding the relationship between gangliosides and various neuronal markers from routinely paraformaldehyde-fixed nervous tissues, both freshly prepared specimens and those stocked in the laboratory.

Novel Targeting to XCR1+ Dendritic Cells Using Allogeneic T Cells for Polytopical Antibody Responses in the Lymph Nodes.

Vaccination strategy that induce efficient antibody responses polytopically in most lymph nodes (LNs) against infections has not been established yet. Because donor-specific blood transfusion induces anti-donor class I MHC antibody production in splenectomized rats, we examined the mechanism and significance of this response. Among the donor blood components, T cells were the most efficient immunogens, inducing recipient T cell and B cell proliferative responses not only in the spleen, but also in the peripheral and gut LNs. Donor T cells soon migrated to the splenic T cell area and the LNs, with a temporary significant increase in recipient NK cells. XCR1+ resident dendritic cells (DCs), but not XCR1- DCs, selectively phagocytosed donor class I MHC+ fragments after 1 day. After 1.5 days, both DC subsets formed clusters with recipient CD4+ T cells, which proliferated within these clusters. Inhibition of donor T cell migration or depletion of NK cells by pretreatment with pertussis toxin or anti-asialoGM1 antibody, respectively, significantly suppressed DC phagocytosis and subsequent immune responses. Three allogeneic strains with different NK activities had the same response but with different intensity. Donor T cell proliferation was not required, indicating that the graft vs. host reaction is dispensable. Intravenous transfer of antigen-labeled and mitotic inhibitor-treated allogeneic, but not syngeneic, T cells induced a polytopical antibody response to labeled antigens in the LNs of splenectomized rats. These results demonstrate a novel mechanism of alloresponses polytopically in the secondary lymphoid organs (SLOs) induced by allogeneic T cells. Donor T cells behave as self-migratory antigen ferries to be delivered to resident XCR1+ DCs with negligible commitment of migratory DCs. Allogeneic T cells may be clinically applicable as vaccine vectors for polytopical prophylactic antibody production even in asplenic or hyposplenic individuals.

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. METHODS: Thoracic duct lymphocytes of congeneic rats were intravenously transferred to host rats and donor cell trafficking in the liver and hepatic lymph was examined. Host hepatic lymph nodes (HLNs) were selectively removed, which allowed liver-derived donor cells to collect in the thoracic duct without transit in the intervening HLNs. RESULTS: The number of donor cells in the thoracic duct lymph significantly increased over the baseline 3, 5 and 11 h after transfer in the HLN-removed, non-pretreated, and HLN-ligated (in which a lymph efflux was blocked) groups, respectively. Histologically, donor cells appeared in the portal area from 0.5 h after transfer and frequently attached to the basal lamina of portal vein both externally and internally. Three hours after transfer, a few donor cells appeared in the subcapsular sinus of HLNs. CONCLUSION: The minimal transit time of rat recirculating lymphocytes is 3-4 h in the liver and 5-8 h in the hepatic LNs, in a normal steady state. Recirculating lymphocytes might transmigrate through the portal vein as well as the sinusoid in the periportal zone. This rapid transit might enable an efficient surveillance of the liver portal area by the recirculating lymphocytes.