Graft-infiltrating Lymphocytes Research Articles

Early graft-infiltrating lymphocytes are not associated with graft rejection in a mouse model of skin transplantation.

Graft-infiltrating lymphocytes (GILs) play an important role in promoting rejection after organ transplantation. We recently reported that GILs that accumulated up to 3 days post-transplantation did not promote rejection, whereas GILs present 3-5 days post-transplantation promoted rejection in a mouse heart transplantation model. However, the immunological behaviour of GILs in murine skin transplantation remains unclear. GILs were isolated on days 3, 5 or 7 post-transplantation from C57BL/6 (B6) allogeneic skin grafts transplanted onto BALB/c mice. BALB/c Rag2-/- γc-/- mice (BRGs) underwent B6 skin graft transplantation 10 weeks after adoptive transfer of day 3, 5, or 7 GILs. BRGs reconstituted with day 5 or 7 GILs completely rejected B6 grafts. However, when B6 grafts harvested from recipient BALB/c mice on day 5 or 7 were re-transplanted into BRGs, half of the re-transplanted day 5 grafts established long-term survival, although all re-transplanted day 7 grafts were rejected. BRGs reconstituted with day 3 GILs did not reject B6 grafts. Consistently, re-transplantation using day 3 skin grafts resulted in no rejection. Administration of anti-CD25 antibodies did not prevent the phenomenon observed for the day 3 skin grafts. Furthermore, BRGs reconstituted with splenocytes from naïve BALB/c mice immediately rejected the naïve B6 skin grafts and the re-transplanted day 3 B6 grafts, suggesting that day 3 GILs were unable to induce allograft rejection during the rejection process. In conclusion, the immunological role of GILs depends on the time since transplantation. Day 3 GILs had neither protective nor alloreactive effects in the skin transplant model.

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+ Tcell ratio remained low, even in the tolerance phase. Furthermore, a PhenoGraph clustering analysis revealed that exhausted CD8+ Tcells 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.

Functional roles of graft‐infiltrating lymphocytes during early‐phase post‐transplantation in mouse cardiac transplantation models

Immunological behavior of graft-infiltrating lymphocytes (GILs) determines the graft fate (i.e., rejection or acceptance). Nevertheless, the functional alloreactivity and the phenotype of GILs at various times during the early post-transplantation phase have not been fully elucidated. We examined the immunological activities of early-phase GILs using a murine model of cardiac transplantation. GILs from 120-h allografts, but not 72-h allografts, showed robust activation and produced proinflammatory cytokines. In particular, a significant increase in CD69+ T-bet+ Nur77+ T cells was detected in 120-h allografts. Furthermore, isolated GILs were used to reconstitute BALB/c Rag2-/- γc-/- (BRG) mice. BRG mice reconstituted with 120-h GILs displayed donor-specific immune reactivity and rejected donor strain cardiac allografts; conversely, 72-h GILs exhibited weak anti-donor reactivity and did not reject allografts. These findings were confirmed by re-transplantation of cardiac allografts into BRG mice at 72-h post-transplantation. Re-transplanted allografts continued to function for >100 days, despite the presence of CD3+ GILs. In conclusion, the immunological behavior of GILs considerably differs over time during the early post-transplantation phase. A better understanding of the functional role of early-phase GILs may clarify the fate determination process in the graft-site microenvironment.

PRESENCE OF GRAFT-INFILTRATING REGULATORY T CELLS ARE ASSOCIATED WITH LONG TERM CARDIAC XENOGRAFT SURVIVAL IN NON-HUMAN PRIMATE

Introduction: Regulatory T (CD4+CD25HiFoxP3+) cells (Treg) are a very small subset of CD4+ T cells that have been shown to play an important role in immune regulation and induce tolerance in transplantation. Previously we have reported the increased number of Treg cells in peripheral blood of long term cardiac xenograft survival recipients. However, in both kidney and liver transplantation in humans, FoxP3+ Tregs have also been associated with clinical rejection. Therefore, the role and function of graft infiltrating Tregs have been of great interest. In this study, we have examined the FoxP3+ Tregs in graft-infiltrating lymphocytes (GILS) from cardiac xenograft. Materials and Methods: Genetically engineered (GE) donor pig heart was heterotopically transplanted in 3-4-year-old specific pathogen-free baboon (n=6). These GE pigs were alpha 1-3 galactosidase knockout and had multiple combinations of transgenic expression of human complement inhibitory protein (CD46), thrombomodulin (TBM), endothelial protein C receptor (EPCR), tissue factor pathway inhibitor (TFPI), and decay-accelerating factor (DAF) and protective gene against phagocytosis (CD47). Immunosuppression consisted of targeted T and B cell depletion and conventional anti-rejection agents. Immuno-phenotyping on GILS from explanted xenograft was performed to analyze the percentage of Treg cells with anti-human CD3, CD4, CD25, CD127, FoxP3, and Helios monoclonal antibodies. Results and Discussion: Cardiac xenotransplantation were performed without complication. Baboons were extubated immediately following surgery and were active, eating, and generally well soon after. WBC and lymphocytes counts were low in recipients after the xenotransplantation due to immunosuppression but recovered in a few days to a normal level. Three of six cardiac xenografts were explanted after 115 days of cardiac xenotransplantation and GILS were examined. A small population of lymphocytes was seen in all the explanted xenograft and CD4+CD25HiFoxP3+ Tregs were examined. Conclusion: We found that long term survivors had an increased percentage of FoxP3+ Treg cells compared to the GILS in cardiac xenografts with earlier graft failure. Tregs were not identified in GILS from recipients with early graft failure. The presence of Tregs was associated with improved graft function. This suggests a TReg-dependent mechanism of survival for xenografts and supports future experiments investigating this mechanism to bring xenotransplantation to clinical reality.

LIVER TISSUE-RESIDENT LYMPHOCYTES ARE SIGNIFICANTLY HETEROGENEOUS, AND THE ORIGINAL REPERTOIRE IS NOT RECREATED BY INFILTRATING RECIPIENT CELLS

Introduction: Tissue-resident (TR) lymphocytes play an unknown but likely significant role in the outcome of solid organ transplantation due to their localisation and low activation threshold. Phenotypic identification of TR cells is limited by organ-specific protein expression and the perhaps incorrect presumption of subset homogeneity. The definition and destiny of these lymphocytes after transplantation has not previously been shown. The liver is the ideal organ in which to study TR lymphocytes due to the abundance of all currently known populations. Methods: Orthotopic liver transplants were performed between MHC matched or mismatched donor and recipient mice. No immunosuppressant drugs were administered to any animal. Graft rejection was assessed by an experienced histopathologist. Donor- and recipient-derived lymphocytes were detected and analysed from grafted livers, bone marrow (BM), spleen, lymph nodes (LN) and blood by flow cytometry. Results: Each lymphocyte subset analysed contained a proportion of TR cells that were retained long-term after MHC matched transplantation. Surprisingly, TR T cells were largely heterogeneous in phenotype, variability which has not previously been appreciated. After MHC mismatched transplantation all donor-derived TR lymphocytes were deleted. Analysis of recipient-derived graft-infiltrating lymphocytes revealed nil to limited proportions of these cells gained a TR phenotype, instead continuing to circulate through the graft. Conclusion: We have described in detail the retention and phenotypic profile of TR lymphocytes after liver transplantation. Despite loss of some to all TR lymphocytes, infiltrating recipient-derived lymphocytes do not recreate the original TR population repertoire. The ability of the graft to fight infection may therefore be hindered by the lack of these first line defenders. Charlies Foundation for Research. Sir Charles Gairdner Osborne Park Health Care Group Research Advisory Committee.

C5aR1 regulates migration of suppressive myeloid cells required for costimulatory blockade-induced murine allograft survival.

Costimulatory blockade-induced murine cardiac allograft survival requires intragraft accumulation of CD11b+ Ly6Clo Ly6G- regulatory myeloid cells (Mregs) that expand regulatory T cells (Tregs) and suppress effector T cells (Teffs). We previously showed that C5a receptor (C5aR1) signaling on T cells activates Teffs and inhibits Tregs, but whether and/or how C5aR1 affects Mregs required for transplant survival is unknown. Although BALB/c hearts survived >60days in anti-CD154 (MR1)-treated or cytotoxic T-lymphocyte associated protein 4 (CTLA4)-Ig-treated wild-type (WT) recipients, they were rejected at ~30days in MR1-treated or CTLA4-Ig-treated recipients selectively deficient in C5aR1 restricted to myeloid cells (C5ar1fl/fl xLysM-Cre). This accelerated rejection was associated with ~2-fold more donor-reactive T cells and ~40% less expansion of donor-reactive Tregs. Analysis of graft-infiltrating mononuclear cells on posttransplant day 6 revealed fewer Ly6Clo monocytes in C5ar1fl/fl xLysM-Cre recipients. Expression profiling of intragraft Ly6Clo monocytes showed that C5aR1 deficiency downregulated genes related to migration/locomotion without changes in genes associated with suppressive function. Cotransfer of C5ar1fl/fl and C5ar1fl/fl xLysM-Cre myeloid cells into MR1-treated allograft recipients resulted in less accumulation of C5ar1-/- cells within the allografts, and in vitro assays confirmed that Ly6Chi myeloid cells migrate to C5a/C5aR1-initiated signals. Together, our results newly link myeloid cell-expressed C5aR1 to intragraft accumulation of myeloid cells required for prolongation of heart transplant survival induced by costimulatory blockade.

Role of miR-449a in the Activation and Metabolism of CD4+ T Cells

BackgroundAcute rejection is a significant challenge after organ transplantation. The CD4+ T-cell‒mediated immune response plays an important role in acute transplant rejection. It was also found that miR-449a microRNA regulates the alloimmune response in a model of heart transplantation in mice. Our goal was to determine the role of miR-449a in the regulation of CD4+ T cells. MethodsWe examined miR-449a expression in peripheral blood mononuclear cells (PBMCs) and graft-infiltrating lymphocytes (GILs) between syngeneic transplant and allogeneic transplant groups on day 7 post‒heart transplantation. We also examined miR-449a expression in CD4+ T-cell activation and mixed-lymphocyte reactions (MLRs) in vitro. To evaluate the effect of miR-449a on CD4+ T-cell metabolism, we analyzed key metabolic parameters using XFp extracellular flux analyses. ResultsOur in vivo heart transplant models showed that the expression of miR-449a in PBMCs and in GILs significantly increased in the allogeneic groups in comparison to the syngeneic groups (P < .01). Furthermore, in vitro analysis confirmed that the expression of miR-449a was significantly elevated in activated CD4+ T cells. Reduction of miR-449a expression in CD4+ T cells decreased the mitochondrial respiration in the same CD4+ T cells. ConclusionOur results reveal that miR-449a microRNA was elevated in allogeneic heart allografts. This correlated with an increased miR-449a expression in activated CD4+ T cells. Inhibition of miR-449a in activated CD4+ T cells coincided with reduced mitochondrial respiration, suggesting that miR-449a influences CD4+ T-cell activation during the alloimmune response by regulating metabolic status.

Alloimmune Responses of Humanized Mice to Human Pluripotent Stem Cell Therapeutics

SummaryThere is growing interest in using embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an “allogenized” mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts.

Differential Expression of Long Noncoding RNAs During Cardiac Allograft Rejection.

Organ transplantation is the most effective treatment for end-stage diseases. Although transplant rejection is the major restriction in successful long-lasting graft survival, induction of sustainable immune tolerance against transplant is one of the major goals in transplantation to enable long-term graft survival. Although various mechanisms have been suggested that induce immune tolerance during transplantation, the roles of long noncoding RNAs (lncRNAs), which modulate gene expression and regulate innate and adaptive immune responses, are not clearly understood in transplantation. Here, we described the role of 2 essential lncRNAs, lncRNA-A930015D03Rik and mouselincRNA1055, in regulating T helper 1 (Th1) response in graft rejection. To understand the gene expression and lncRNA profile during transplantation, we performed microarray to profile lncRNA and messenger (m)RNA of the heart graft and graft-infiltrating lymphocytes (GILs) in allogeneic and syngeneic mouse heart transplantation model. We screened the differentially expressed lncRNAs and mRNAs, and generated the network of lncRNA-mRNA coexpression and computationally predicted their association in transplantation. We further validated the selected T cell related lncRNAs by qPCR, which we identified in gene set enrichment analysis. The functional validation of these lncRNAs in the regulation of Th1 response in transplantation was performed by short hairpin RNA-mediated inhibition. We established a profile of lncRNA and mRNA, which are differentially expressed during transplant rejection in mouse model of heart transplant. Consistent with the microarray results, we have confirmed and validated the expression of 7 lncRNA by qPCR. The lncRNA-A930015D03Rik and mouselincRNA1055 were highly expressed in allogeneic heart graft and GILs. We further identified that expression of IL-12Rβ1 is strongly correlated with lncRNA-A930015D03Rik and mouselincRNA1055 in GILs. Further analysis revealed the association of lncRNA-A930015D03Rik and mouselincRNA1055 with Th1 cells in graft rejection. The functions of lncRNA-A930015D03Rik and mouselincRNA1055 were validated in differentiation of Th1 cells by knocking down their expression. Inhibition of lncRNA-A930015D03Rik and mouselincRNA1055 substantially suppressed the expression of IL-12Rβ1 and IFN-γ induction in Th1 cells. Our results provide a detailed profile of lncRNAs that may regulate immune response and graft outcomes. Our data not only suggest the involvement of lncRNA-A930015D03Rik and mouselincRNA1055 in the regulation of Th1 cells response during graft rejection but also identify them as novel biomarkers for subclinical graft rejection.

Anti-OX40L monoclonal antibody prolongs secondary heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade

BackgroundMemory T cells (Tms) form a barrier against long-term allograft survival; however, CD4+Foxp3+ regulatory T cells (Tregs) can suppress allograft rejection. The OX40/OX40L pathway is critical to the generation of Tms and turns off Treg suppressor function. MethodsB6 mice that rejected BALB/c skin grafts after 4weeks were used as the secondary heart transplant recipients. The skin recipient mice, termed S0, S2 and S3, were treated with the isotype antibodies, anti-CD40L/LFA-1 or anti-OX40L combined with anti-CD40L/LFA-1 mAbs, respectively. The secondary heart recipients, termed H0 and H2, received anti-CD40L/LFA-1 mAbs or not, respectively (Fig. 1). ResultsFour weeks after primary skin transplantation, the Tms in the S3 group that received anti-OX40L with anti-CD40L/LFA-1 mAbs were reduced compared to those in the S2 group (CD4+ Tm: 32.61±2.20% in S2 vs. 25.36±1.16% in S3; CD8+ Tm: 27.76±1.96% in S2 vs. 20.95±1.30% in S3; P<0.01). Meanwhile, the proportions of Tregs in S3 increased compared to those in S2 (P<0.05). The anti-OX40L with anti-CD40L/LFA-1 mAbs group (S3H2) prolonged the mean survival time (MST) following secondary heart transplantation from 9.5days to 21days (P<0.001). Furthermore, allogeneic proliferation of recipient splenic T cells and graft-infiltrating lymphocytes were significantly inhibited in the S3H2 group. Additionally, a higher level of IL-10 was detected in sera and allografts. ConclusionsAnti-OX40L mAb could prolong secondary heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade. The mechanism of protecting allografts using anti-OX40L mAb involved impairing the generation of Tm and up-regulating IL-10 producing Tregs, inhibiting the function of T cells.

Interleukin-17–producing CD4+ cells home to the graft early after human heart transplantation

Interleukin-17 (IL-17) is regarded as a major effector cytokine with pro-inflammatory actions. It has pleiotropic and environment-specific functions by promoting adaptive cytotoxic T-lymphocyte responses during inflammation. Therefore, it is tempting to speculate that IL-17 plays a major role in inflammatory responses in transplant recipients. We questioned whether IL-17 is expressed in the transplanted heart during acute rejection (AR), or during immunologic quiescence, and which graft-infiltrating lymphocytes produce IL-17. In addition, we analyzed donor-specific IL-17-producing cells in peripheral blood cells in comparable periods after transplantation. Endomyocardial biopsies from heart transplant recipients with early or late AR or in an immunologic quiescence period were analyzed for the presence of IL-17 mRNA. In addition, the capacity of graft-infiltrating lymphocytes (GILs) to produce IL-17 was analyzed. Moreover, we determined the frequency of donor-reactive IL-17-producing peripheral blood mononuclear cells (PBMCs) using an Elispot assay. Twenty-one percent (14 of 67) of the biopsies assessed were positive for IL-17 mRNA. Thirteen of 41 biopsies were observed in the early period (≤3 months) after transplantation. One (of 26) of the late biopsies expressed IL-17 (p = 0.006). Specifically, IL-17 was expressed during early AR (57%, or 8 of 14), whereas biopsies from late AR (0 of 5) did not express IL-17 mRNA (p = 0.02). During AR, IL-17 is derived from IL-17-producing CD4(+)CD161(+), and not CD8(+), GILs. In contrast to the graft findings, we detected circulating donor-reactive IL-17-producing cells mostly during immunologic quiescence. Particularly early after heart transplantation, IL-17-producing CD4(+) T cells home to the graft, which contributes to the AR process.

IL-17 Producing CD4+ Cells Infiltrate the Graft During Early Rejection Episodes.

IL-17 is regarded as a major effector cytokine with pro-inflammatory actions in autoimmune diseases. IL-17 has pleiotropic and environmental specific functions by promoting adaptive cytotoxic T-lymphocyte responses during inflammation. Therefore, it is tempting to speculate that IL-17 is important in inflammatory responses seen after organ transplantation. We determined IL-17 mRNA expression in the transplanted heart during early and late acute rejection episodes, and during immunological quiescence. Additionally, we analysed whether IL-17 is derived from graft-infiltrating lymphocytes. Endomyocardial biopsies (EMBs: n=41) from heart transplant recipients (n=29) who experienced an early (≤3 months) or late (>3 months) acute rejection episode or during an immunological quiescence period, were analysed for the presence of IL-17 mRNA. IL-17 production determined by flowcytometry was analysed in cultures of graft-infiltrating lymphocytes (GILs) derived from acute rejection (n=5) and non-rejection (n=5) EMBs stimulated with PMA and ionomycine. Twenty-two percent (9/41) of EMBs were positive for IL-17 mRNA. All (9/26) were observed in the early period after transplantation, while none (0/15) of the late biopsies expressed IL-17 mRNA (p=0.02). During early acute rejection, 56% (5/9) of the EMBs did express IL-17 mRNA, while EMBs from late acute rejections (0/5) did not express IL-17 mRNA (p=0.09). Only CD4+ cells from GIL cultures produced IL-17. In 60% of GIL cultures from acute rejection EMBs a significant proportion of CD4+ cells produced IL-17, while only one GIL culture from non-rejection EMBs contained CD4+IL-17+ cells. All CD4+IL-17+ cells expressed CD161, a cell surface expression marker of Th17 cells. CD8+ cells did not produce IL-17 neither expressed CD161. In conclusion, particularly early after heart transplantation IL-17 producing CD4+ T-cells home to the graft contributing to the acute rejection process.

MicroRNA-155 regulates T cell proliferation through targeting GSK3β in cardiac allograft rejection in a murine transplantation model

Here we investigated the activity and regulation of miR-155 during cardiac allograft rejection (AR), and to examine the feasibility of using miR-155 as a biomarker of graft status. Expression of miR-155 in graft-infiltrating lymphocytes (GIL), T cells isolated from spleen (TFS), and lymphocytes separated from blood (LFB) was significantly increased during cardiac AR while GSK3β was downregulated in GIL and TFS. Inhibition of miR-155 impaired lymphocyte proliferation and enhanced the expression of GSK3β. Moreover, pharmacological inactivation of GSK3β resulted in rescue of the proliferative capability of T cells pretreated with a miR-155 inhibitor. Luciferase reporter assay confirmed that miR-155 interacted with the 3′-untranslated region (UTR) of GSK3β directly. In particular, the miR-155 in LFB can distinguish recipients with AR from syngeneic controls from POD 3 and later. The present study provides a better understanding of the pathophysiological process underlying cardiac AR progression.

Long-Term Heart Transplant Survival by Targeting the Ionotropic Purinergic Receptor P2X7

Heart transplantation is a lifesaving procedure for patients with end-stage heart failure. Despite much effort and advances in the field, current immunosuppressive regimens are still associated with poor long-term cardiac allograft outcomes, and with the development of complications, including infections and malignancies, as well. The development of a novel, short-term, and effective immunomodulatory protocol will thus be an important achievement. The purine ATP, released during cell damage/activation, is sensed by the ionotropic purinergic receptor P2X7 (P2X7R) on lymphocytes and regulates T-cell activation. Novel clinical-grade P2X7R inhibitors are available, rendering the targeting of P2X7R a potential therapy in cardiac transplantation. We analyzed P2X7R expression in patients and mice and P2X7R targeting in murine recipients in the context of cardiac transplantation. Our data demonstrate that P2X7R is specifically upregulated in graft-infiltrating lymphocytes in cardiac-transplanted humans and mice. Short-term P2X7R targeting with periodate-oxidized ATP promotes long-term cardiac transplant survival in 80% of murine recipients of a fully mismatched allograft. Long-term survival of cardiac transplants was associated with reduced T-cell activation, T-helper cell 1/T-helper cell 17 differentiation, and inhibition of STAT3 phosphorylation in T cells, thus leading to a reduced transplant infiltrate and coronaropathy. In vitro genetic upregulation of the P2X7R pathway was also shown to stimulate T-helper cell 1/T-helper cell 17 cell generation. Finally, P2X7R targeting halted the progression of coronaropathy in a murine model of chronic rejection as well. P2X7R targeting is a novel clinically relevant strategy to prolong cardiac transplant survival.

MicroRNA-182 is a key modulator of FOXO1 expression and induced in response to alloantigens during acute rejection

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate the post-transcriptional expression of target genes and have been shown to be important regulators of many biologic processes including innate and adaptive immune responses. The roles of specific miRNAs in alloimmune responses remain unknown. We used a mouse model of heart transplantation and microarray analysis to profile miRNAs differentially expressed during allograft rejection with the goal of identifying miRNAs that regulate alloimmune responses and graft outcomes. Syngeneic (C57BL/6 → C57BL/6) and allogeneic (BALB/c → C57BL/6) (n=3-9) heterotopic heart transplants were performed. Heart grafts were collected on days 1-7 and graft infiltrating lymphocytes (GILs) were isolated on day 5 post-transplant. Samples were profiled using TaqMan microfluidic cards that contain 335 mature mouse miRNAs. Microarray data were analyzed to define miRNAs that are differentially expressed (P< 0.05). Differentially expressed miRNAs were validated by Taqman miRNA assays using samples from additional groups (n=3-5) of transplanted mice. Sixteen miRNAs were significantly differentially expressed in both the heart grafts and the GILs of allogeneic recipients, compared to syngeneic recipients, including the miRNA miR-182, a dominantly expressed member of miR-183-96-182 cluster. Forkhead Box (FOX) proteins are a family of important transcription factors and FOXO1 is a predicted target of miR-182. We used a luciferase reporter assay system and confirmed FOXO1 is a target of miR-182 as mutation of the Foxo1 seed sequence decreased specific binding to miR-182. FOXO1 expression in grafts was analyzed by qPCR and western blot on days 1-5 post-transplant and immunohistochemistry was used to detect FOXO1 expression in cardiomyocytes and GILs. We show that as miR-182 increases after transplant, there is a concomitant post-transcriptional decrease in FOXO1 expression in heart allografts. Further FOXO1 expression is localized to both the cardiomyocytes and CD3+ T cells. Importantly, miR-182 was significantly increased in both the PBLs and plasma from recipients of allogeneic grafts as compared to recipients of syngeneic grafts and normal mice. These data indicate that miR-182 expression is specifically expressed in the circulation of graft recipients and suggest that since miRNAs are incredibly stable in blood, they will be important as biomarkers of graft status. Our results suggest that miRNAs may have important regulatory functions post-transplant.

Effects of a calcineurin inhibitor, FK506, and a CCR5/CXCR3 antagonist, TAK-779, in a rat small intestinal transplantation model

The effects of FK506, and TAK-779, antagonists of CCR5 and CXCR3, were investigated using a rat intestinal transplantation model. Small intestines from DA rats were heterotopically transplanted into LEW rats. The recipients were treated with FK506 (1 mg/kg/day, day 0–5) and TAK-779 (10 mg/kg/day, day 0–10). Graft survival and immunological responses to these materials were estimated by mixed lymphocyte reactions and IFN-γ production. The expression of chemokine receptors on lymphocytes was also examined. The average duration of survival was 7.0 ± 0.3, 12.0 ± 1.0, 9.8 ± 0.5 and 18.0 ± 1.5 days in the allogeneic, FK506, TAK-779 and the two-drug combined groups, respectively. Cell proliferative responses and IFN-γ production were suppressed to a significant extent in the FK506 group compared with the TAK-779 group. In addition, the two-drug combination showed a tendency for stronger suppression than FK506 alone, correlated with in vivo and histopathological data. The numbers of both CD4 + and CD8 + cells were significantly suppressed in the blood of the recipients of both the FK506 and the TAK-779 groups, and in Peyer's patches of the graft of the TAK-779 group, but the FK506 group was not, as evidenced by FACS analysis. In addition, double-staining of graft-infiltrating lymphocytes showed a significant reduction in lymphocyte numbers, expressing CCR5 and CXCR3 in the TAK-779 group, but not evident in the FK506 group, compared to the allogeneic group. While FK506 suppresses cell proliferation and effecter function, it has less effect on the expression of CCR5 and CXCR3 in lymphocytes. Further exploration of the effects of a combined therapy with TAK-779 could represent a novel treatment for intestinal transplantation.

Acute rejection results in a unique signature of microRNAs (169.22)

Abstract MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate the post-transcriptional expression of genes. Little is known about the roles of specific miRNAs in alloimmune responses. Groups of syngeneic and allogeneic recipients of heart transplants were sacrificed on day 7 post-transplant and heart grafts and spleens obtained. Graft infiltrating lymphocytes (GIL) and peripheral blood lymphocytes (PBL) were isolated on post-transplant day 5. MicroRNAS were profiled using TaqMan microfluidic cards. Forty-two miRNAs were increased and 32 were decreased (p&amp;lt;0.05) in allogeneic heart grafts as compared to syngeneic. To determine if these miRNAs were expressed by lymphocytes or heart tissue, we profiled the GIL and determined that 22 miRNAs were increased and 52 decreased. There was less differential expression seen in spleen and PBL with 11 and 9 miRNAs changed respectively. Seventeen miRNAs were selected for validation by qPCR. The miRNAs, miR-7a and miR-182 were significantly increased in the heart graft, GIL, and PBL indicating that acute rejection results in specific profile of miRNAs in both the graft and the periphery. Further studies analyzing the targets of the miRNAs associated with graft rejection may lead to novel therapeutics and diagnostics.

Blockade of CD27/CD70 pathway to reduce the generation of memory T cells and markedly prolong the survival of heart allografts in presensitized mice

Background Alloreactive memory T cells are a major obstacle to transplantation acceptance due to their capacity for accelerated rejection. Methods C57BL/6 mice that had rejected BALB/c skin grafts 4 weeks earlier were used as recipients. The recipient mice were treated with anti-CD154/LFA-1 with or without anti-CD70 during the primary skin transplantation and anti-CD154/LFA-1 or not during the secondary transplantation of BALB/c heart. We evaluated the impact of combinations of antibody-mediated blockade on the generation of memory T cells and graft survival after fully MHC-mismatched transplantations. Results One month after the primary skin transplantation, the proportions of CD4 + memory T cells/CD4 + T cells and CD8 +memory T cells/CD8 + T cells in the anti-CD154/LFA-1 combination group were 47.32 ± 4.28% and 23.18 ± 2.77%, respectively. In the group that included anti-CD70 treatment, the proportions were reduced to 34.10 ± 2.71% and 12.19 ± 3.52% (P < 0.05 when comparing the proportion of memory T cells between the two groups). The addition of anti-CD70 to the treatment regimen prolonged the mean survival time following secondary heart transplantation from 10 days to more than 90 days (P < 0.001). Furthermore, allogenic proliferation of recipient splenic T cells and graft-infiltrating lymphocytes were significantly decreased. Meanwhile, the proportion of regulatory T cells was increased to 9.46 ± 1.48% on day 100 post-transplantation (P < 0.05). Conclusions The addition of anti-CD70 to the anti-CD154/LFA-1 combination given during the primary transplantation reduced the generation of memory T cells. This therapy regimen provided a potential means to alleviate the accelerated rejection mediated by memory T cells during secondary heart transplantation and markedly prolong the survival of heart allografts.

Administration of anti-interleukin-6 monoclonal antibody prolongs cardiac allograft survival

To investigate the effects of anti-IL-6 monoclonal antibody (anti-IL-6 mAb) on acute allograft rejection and the potential mechanisms in a mouse heart transplantation model. Heterotopical heart graft model was performed. The anti-IL-6 mAb was administered to recipient mice after cardiac grafting. Results were compared with administration of anti-IL-17 mAb or anti-IL-6 mAb+anti-IL-17 mAb (the 'double' treatment). The cardiac allograft survival was monitored by daily palpation in combination with histological evaluation. Quantitative polymerase chain reaction assay, mixed lymphocyte reaction, and flow cytometric analysis were employed to determine the mRNA expression of pro-inflammatory cytokines, allogeneic T-cell proliferation, and the proportion of CD4(+) CD25(+) Foxp3(+) regulatory T cells in graft-infiltrating lymphocytes and splenocytes of recipients, respectively. The results showed that the cardiac allograft survival in anti-IL-6 mAb-treated mice was prolonged significantly when compared with that of the untreated or anti-IL-17 mAb-treated mice. Meanwhile, the 'double-treated' did not prolong graft survival significantly when compared with those treated with anti-IL-6 mAb. The increase of graft survival induced by anti-IL-6 mAb was associated with reduced transcript levels for IFN-γ and IL-17, accompanied by a dramatic reduction of T-cell proliferation capacity to alloantigen stimuli and a higher proportion of Treg cells. Thus, anti-IL-6 mAb may be protective against acute rejection after cardiac transplantation through suppressing the activation of effector T cells and promoting the induction of Treg cells.

Induction of Lymphocyte Apoptosis in Rat Liver Allograft by Adenoviral Gene Transfection of Human Interleukin-10

Background/Aims: Gene therapy can provide a possible avenue in organ transplantation to treat acute allograft rejection. This study was designed to investigate the effect of adenovirus-mediated human IL-10 (hIL-10) gene transfer on the apoptosis of infiltrating lymphocytes and examine the efficacy of hIL-10 gene transfer in combination with subtherapeutic doses of cyclosporine A (CsA) in a rat liver transplantation model. Methods: Inbred male DA and LEW rats were used for liver donors and recipients, respectively. The rats were divided into saline, Ad-lacZ, CsA, Ad-hIL-10 and Ad-hIL-10 + CsA groups. Graft survival, histopathological, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction and flow cytometry were performed in liver specimens obtained from different time points after transplantation in the 5 groups. Results: Ad-hIL-10 pretreatment inhibited allograft rejection, prolonged the survival of hepatic allografts, and downregulated the expression of IFN-γ and IL-2 mRNA, with simultaneous upregulation of IL-4 mRNA. In addition, Ad-hIL-10 pretreatment upregulated the expression of Fas mRNA in the isolated graft-infiltrating lymphocytes and induced graft-infiltrating lymphocyte apoptosis. A single subtherapeutic dose of CsA acted synergistically with it. Conclusion: hIL-10 gene therapy induced alloreactive lymphocyte apoptosis via Fas/FasL pathway. hIL-10 gene transfection in combination with subtherapeutic doses of CsA facilitates the long-term survival of liver grafts.