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.

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.

Estimating illuminant color based on luminance balance of surfaces.

To accomplish color constancy the illuminant color needs to be discounted from the light reflected from surfaces. Some strategies for discounting the illuminant color use statistics of luminance and chromaticity distribution in natural scenes. In this study we showed whether color constancy exploits the potential cue that was provided by the luminance balance of differently colored surfaces. In our experiments we used six colors: bright and dim red, green, and blue, as surrounding stimulus colors. In most cases, bright colors were set to be optimal colors. They were arranged among 60 hexagonal elements in close-packed structure. The center element served as the test stimulus. The observer adjusted the chromaticity of the test stimulus to obtain a perceptually achromatic surface. We used simulated black body radiations of 3000 (or 4000), 6500, and 20000 K as test illuminants. The results showed that the luminance balance of surfaces with no chromaticity shift had clear effects on the observer's achromatic setting, which was consistent with our hypothesis on estimating the scene illuminant based on optimal colors.

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.

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.

High-endothelial cell-derived S1P regulates dendritic cell localization and vascular integrity in the lymph node.

While the sphingosine-1-phosphate (S1P)/sphingosine-1-phosphate receptor-1 (S1PR1) axis is critically important for lymphocyte egress from lymphoid organs, S1PR1-activation also occurs in vascular endothelial cells (ECs), including those of the high-endothelial venules (HEVs) that mediate lymphocyte immigration into lymph nodes (LNs). To understand the functional significance of the S1P/S1PR1-Gi axis in HEVs, we generated Lyve1;Spns2Δ/Δ conditional knockout mice for the S1P-transporter Spinster-homologue-2 (SPNS2), as HEVs express LYVE1 during development. In these mice HEVs appeared apoptotic and were severely impaired in function, morphology and size; leading to markedly hypotrophic peripheral LNs. Dendritic cells (DCs) were unable to interact with HEVs, which was also observed in Cdh5CRE-ERT2;S1pr1Δ/Δ mice and wildtype mice treated with S1PR1-antagonists. Wildtype HEVs treated with S1PR1-antagonists in vitro and Lyve1-deficient HEVs show severely reduced release of the DC-chemoattractant CCL21 in vivo. Together, our results reveal that EC-derived S1P warrants HEV-integrity through autocrine control of S1PR1-Gi signaling, and facilitates concomitant HEV-DC interactions.

Involvement of the programmed death-1/programmed death-1 ligand pathway in CD4+CD25+ regulatory T-cell activity to suppress alloimmune responses.

BACKGROUND: Immune regulatory CD4+CD25+ T (regulatory T; Treg) cells play a vital role in the induction and maintenance of self-tolerance. They are essential for the homeostasis of T cells, the prevention of autoimmunity, and the induction of tolerance to allogeneic donor grafts. However, the underlying mechanism of their functions remains mostly elusive. Therefore, we investigated here a crucial role of Treg cells in their response to alloantigen via the programmed death (PD)-1/PD-1 ligand (PD-L1) pathway. METHODS: In vitro mixed lymphocyte reaction (MLR) assay, graft-versus-host disease (GvHD) and a skin transplantation model were used to evaluate the mechanisms of PD-1/PD-L1 pathway. RESULTS: Blockade of the PD-1/PD-L1 pathway using anti-PD-L1 monoclonal antibodies (mAb) is found to inhibit Treg cell's ability to suppress and restore CD4+CD25-T-cell proliferation in vitro. GvHD was lethal after adoptive transfer of allogeneic C57BL/6 (H-2K) spleen cells to NOD/SCID (H-2K) mice unless CD25+ T cells were also included. Strikingly, the suppression of GvHD by CD25+ cells was abrogated by anti-PD-L1 mAb administration. The abrogation of Treg-cell-mediated suppression could also be demonstrated in a Balb/c (H-2K) to B6/Rag-2KO (H-2K) skin-allograft model. CONCLUSIONS: The blockade of the PD-1/PD-L1 pathway abrogates Treg-mediated immunoregulation, thus suggesting that the PD-1/PD-L1 pathway is required for Treg suppression of the alloreactive responses of CD4+CD25-T cells. This finding has important implications for clarifying the mechanisms of allograft rejection and GvHD.

Gene expression profile analysis of the peripheral blood mononuclear cells from tolerant living-donor liver transplant recipients.

Induction of transplant tolerance is a clinically desirable goal. To provide unbiased insight into transplant tolerance, we analyzed gene expression profiling in peripheral blood mononuclear cells from recipients of living-donor liver transplants (LDLTs) who had retained an immune tolerance with a well-functioning graft for several years using cDNA microarray. The comparative analyses with nontransplanted normal healthy volunteers showed that the majority of reliable detected genes were similar, and 5.6% of the genes in the tested genome (of which 627 up-regulated and 90 down-regulated) were significantly regulated and specific to tolerant LDLT recipients, indicating a significant genetic feature for inducing and maintaining immune tolerance. Moreover, the expression of several selected genes was confirmed by semiquantitative reverse transcriptase-polymerase chain reaction, which correlated to microarray data. Our data indicated that cDNA microarray technology was useful for this application and provided many informative insights into transplant tolerance mechanism.

Anergic lymphocytes generated by blocking CD28 and ICOS pathways in vitro prolong rat cardiac graft survival.

Regulatory cells may play a pivotal role in inducing and maintaining transplantation tolerance. We investigated the mechanism of anergic lymphocytes with regulatory cell potential generated in vitro by ICOS and CD 28 co-stimulatory blockades as a source of cellular therapy for treating allograft rejection. Anergic lymphocytes were generated by a mixed lymphocyte reaction consisting of DA splenocytes as the stimulator and Lewis splenocytes as the responder in the presence of anti-ICOS mAb and rCTLA-4I g. Immunoregulatory effects of these lymphocytes were evaluated by secondary MLR and using other various stimulations. DA heart was transplanted into 7.5 Gy-irradiated Lewis rat after intravenous administration of these cells and/or Lewis spleen lymphocytes. We observed that these lymphocytes were not only anergic to alloantigen and polyclonal stimulations but also exhibited regulative activity to inhibit the alloreactive T-cell response. Our adoptive transfer studies revealed that irradiated recipients that received both anergic lymphocytes and naIve Lewis lymphocytes had significantly prolonged DA cardiac graft survival (mean 17.5 days) compared with a group that received Lewis lymphocytes alone (mean 10.8 days). Furthermore, some of the recipients accepted the graft indefinitely after receiving anergic lymphocytes alone (>100 days). These results demonstrated that anergic lymphocytes with regulatory activities can be generated through blocking co-stimulatory signals, CD 28 and ICOS, simultaneously in vitro, and may advance a new immunomodulatory strategy for preventing allorejection in organ transplantation.

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

BACKGROUND: Lymphocyte 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. METHODS: A 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. RESULTS: The immunoelectron microscopic analysis clearly showed CD8ß(+) 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 α4ß1 or αLß2 integrins. Immunohistochemistry showed the expression of CXCL10 in donor MHCII(high) cells in the portal tract as well as endothelial walls of PV. CONCLUSIONS: We 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 α4ß1 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.

Simultaneous blockade of co-stimulatory signals, CD28 and ICOS, induced a stable tolerance in rat heart transplantation.

An inducible co-stimulator (ICOS), a recently identified co-stimulatory receptor with a close structural homology of CD28 and CTLA4, is expressed on activated T cells. Anti-ICOS antibody was demonstrated to be effective on prolongation of graft survival after liver transplantation in rats. In this study, we investigated the potency of tolerance induction using the antibody combined with a recombinant adenovirus vector containing CTLA-4Ig cDNA (AdCTLA-4Ig) in rat heart transplantation model. Using a DA-to-Lewis rat heart transplantation model, an anti-rat ICOS antibody and AdCTLA-4Ig were simultaneously administered i.v. into recipients. The tissue specimens from the grafts were removed on various days after transplantation for histological evaluation. Donor-strain skin and heart grafts, and third-party heart allografts were challenged in the recipients with a long-term surviving graft. Splenocytes from the tolerance-induced recipients were used for adoptive transfer study. Anti-ICOS antibody alone did not prolong the survival of heart allograft. AdCTLA-4Ig monotherapy significantly prolonged the survival of heart allograft (Group 4). With a combination of Anti-ICOS antibody and AdCTLA-4Ig, all recipients were resulted in a long-term allograft acceptance for more than 200 days (Group 8). When challenged donor-strain skin grafts in the tolerant rats of Group 4, the skin was rejected, which also lead to a rejection of primary heart allografts. The recipients in Group 8 also rejected donor-strain skin grafts with no rejection of the primary heart grafts. These recipients accepted secondary heart grafts from donor-strain but not third-party. In Group 8 long-term survival recipients showed a high population of CD4+CD25+ regulatory T cell in peripheral blood, and in adoptive transfer study subtraction of these CD4+CD25+ T cells accelerate the rejection of heart graft in secondary irradiated recipients. The present results demonstrated that anti-ICOS antibody combined with AdCTLA-4Ig potently induces a stable immune tolerance after heart allografting in rat, which is mediated by the induction of CD4+CD25+ regulatory T cells. This strategy may be attractive for clinical employment to induce transplantation tolerance.

Identification of microRNAs involved in acute rejection and spontaneous tolerance in murine hepatic allografts.

Graft acceptance without the need for immunosuppressive drugs is the ultimate goal of transplantation therapy. In murine liver transplantation, allografts are accepted across major histocompatibility antigen complex barriers without the use of immunosuppressive drugs and constitute a suitable model for research on immunological rejection and tolerance. MicroRNA (miRNA) has been known to be involved in the immunological responses. In order to identify mRNAs in spontaneous liver allograft tolerance, miRNA expression in hepatic allografts was examined using this transplantation model. According to the graft pathological score and function, miR-146a, 15b, 223, 23a, 27a, 34a and 451 were upregulated compared with the expression observed in the syngeneic grafts. In contrast, miR-101a, 101b and 148a were downregulated. Our results demonstrated the alteration of miRNAs in the allografts and may indicate the role of miRNAs in the induction of tolerance after transplantation. Furthermore, our data suggest that monitoring the graft expression of novel miRNAs may allow clinicians to differentiate between rejection and tolerance. A better understanding of the tolerance inducing mechanism observed in murine hepatic allografts may provide a therapeutic strategy for attenuating allograft rejection.

Three distinct subsets of thymic epithelial cells in rats and mice defined by novel antibodies.

AIM: Thymic epithelial cells (TECs) are thought to play an essential role in T cell development and have been detected mainly in mice using lectin binding and antibodies to keratins. Our aim in the present study was to create a precise map of rat TECs using antibodies to putative markers and novel monoclonal antibodies (i.e., ED 18/19/21 and anti-CD205 antibodies) and compare it with a map from mouse counterparts and that of rat thymic dendritic cells. RESULTS: Rat TECs were subdivided on the basis of phenotype into three subsets; ED18+ED19+/-keratin 5 (K5)+K8+CD205+ class II MHC (MHCII)+ cortical TECs (cTECs), ED18+ED21-K5-K8+Ulex europaeus lectin 1 (UEA-1)+CD205- medullary TECs (mTEC1s), and ED18+ED21+K5+K8dullUEA-1-CD205- medullary TECs (mTEC2s). Thymic nurse cells were defined in cytosmears as an ED18+ED19+/-K5+K8+ subset of cTECs. mTEC1s preferentially expressed MHCII, claudin-3, claudin-4, and autoimmune regulator (AIRE). Use of ED18 and ED21 antibodies revealed three subsets of TECs in mice as well. We also detected two distinct TEC-free areas in the subcapsular cortex and in the medulla. Rat dendritic cells in the cortex were MHCII+CD103+ but negative for TEC markers, including CD205. Those in the medulla were MHCII+CD103+ and CD205+ cells were found only in the TEC-free area. CONCLUSION: Both rats and mice have three TEC subsets with similar phenotypes that can be identified using known markers and new monoclonal antibodies. These findings will facilitate further analysis of TEC subsets and DCs and help to define their roles in thymic selection and in pathological states such as autoimmune disorders.