Xuyan Huang

Inhibitory Feedback Loop Between Tolerogenic Dendritic Cells and Regulatory T Cells in Transplant Tolerance

An active role of T regulatory cells (Treg) and tolerogenic dendritic cells (Tol-DC) is believed important for the induction and maintenance of transplantation tolerance. However, interactions between these cells remain unclear. We induced donor-specific tolerance in a fully MHC-mismatched murine model of cardiac transplantation by simultaneously targeting T cell and DC function using anti-CD45RB mAb and LF 15-0195, a novel analog of the antirejection drug 15-deoxyspergualin, respectively. Increases in splenic Treg and Tol-DC were observed in tolerant recipients as assessed by an increase in CD4+CD25+ T cells and DC with immature phenotype. Both these cell types exerted suppressive effects in MLR. Tol-DC purified from tolerant recipients incubated with naive T cells induced the generation/expansion of CD4+CD25+ Treg. Furthermore, incubation of Treg isolated from tolerant recipients with DC progenitors resulted in the generation of DC with Tol-DC phenotype. Treg and Tol-DC generated in vitro were functional based on their suppressive activity in vitro. These results are consistent with the notion that tolerance induction is associated with a self-maintaining regulatory loop in which Tol-DC induce the generation of Treg from naive T cells and Treg programs the generation of Tol-DC from DC progenitors.

Dendritic Cells Genetically Engineered to Express Fas Ligand Induce Donor-Specific Hyporesponsiveness and Prolong Allograft Survival

Polarization of an immune response toward tolerance or immunity is dictated by the interactions between T cells and dendritic cells (DC), which in turn are modulated by the expression of distinct cell surface molecules, and the cytokine milieu in which these interactions are taking place. Genetic modification of DC with genes coding for specific immunoregulatory cell surface molecules and cytokines offers the potential of inhibiting immune responses by selectively targeting Ag-specific T cells. In this study, the immunomodulatory effects of transfecting murine bone marrow-derived DC with Fas ligand (FasL) were investigated. In this study, we show that FasL transfection of DC markedly augmented their capacity to induce apoptosis of Fas+ cells. FasL-transfected DC inhibited allogeneic MLR in vitro, and induced hyporesponsiveness to alloantigen in vivo. The induction of hyporesponsiveness was Ag specific and was dependent on the interaction between FasL on DC and Fas on T cells. Finally, we show that transfusion of FasL-DC significantly prolonged the survival of fully MHC-mismatched vascularized cardiac allografts. Our findings suggest that DC transduced with FasL may facilitate the development of Ag-specific unresponsiveness for the prevention of organ rejection. Moreover, they highlight the potential of genetically engineering DC to express other genes that affect immune responses.

Immune Modulation by Silencing IL-12 Production in Dendritic Cells Using Small Interfering RNA

RNA interference is a mechanism of posttranscriptional gene silencing that functions in most eukaryotic cells, including human and mouse. Specific gene silencing is mediated by short strands of duplex RNA of ∼21 nt in length (termed small interfering RNA or siRNA) that target the cognate mRNA sequence for degradation. We demonstrate here that RNAi can be used for immune modulation by targeting dendritic cell (DC) gene expression. Transfection of DC with siRNA specific for the IL-12 p35 gene resulted in potent suppression of gene expression and blockade of bioactive IL-12 p70 production without affecting unrelated genes or cellular viability. Inhibition of IL-12 was associated with increased IL-10 production, which endowed the DC with the ability to stimulate production of Th2 cytokines from allogenic T cells in vitro. Furthermore, siRNA-silenced DC lacking IL-12 production were poor allostimulators in MLR. IL-12-silenced and KLH-pulsed DC polarized the immune response toward a Th2 cytokine profile in an Ag-specific manner. These data are the first to demonstrate that RNA interference is a potent and specific tool for modulating DC-mediated immune responses.

LF15-0195 generates tolerogenic dendritic cells by suppression of NF-κB signaling through inhibition of IKK activity

LF15‐0195 (LF) is a potent, less toxic analog of the immunosuppressant 15‐deoxyspergualine, which we previously reported to prevent graft rejection and to induce permanent tolerance in a murine cardiac transplantation model. However, the underlying mechanism of action of LF required elucidation. In this study, dendritic cells (DC) treated with LF before activation with tumor necrosis factor α (TNF‐α)/lipopolysaccharide (LPS) failed to express maturation markers (major histocompatibility complex II, CD40, CD86) and interleukin‐12. LF prevented, in a concentration‐dependent manner, the activation and nuclear translocation of nuclear factor‐κB (NF‐κB) in DC following addition of TNF‐α/LPS. Yet‐activated and active IκB kinases (IKKs) were inhibited in cells pretreated with LF, thereby preventing the phosphorylation of IκB and release of NF‐κB, a key regulator of genes associated with the maturation of DC. LF‐induced inhibition of IKK activity was reversed in a dose‐dependent manner by the overexpression of IKK. The T helper cell type 2 (Th2) differentiation of naïve T cells promoted by LF‐treated DC in vitro correlates with Th2 polarization observed in transplant recipients made tolerant by LF. These data demonstrated that LF‐induced blockade of NF‐κB signaling at the level of IKK promoted the generation of tolerogenic DC that inhibited Th1 polarization and increased Th2 polarization in vitro and in vivo.

Double-Negative T Cells, Activated by Xenoantigen, Lyse Autologous B and T Cells Using a Perforin/Granzyme-Dependent, Fas-Fas Ligand-Independent Pathway

The ability to control the response of B cells is of particular interest in xenotransplantation as Ab-mediated hyperacute and acute xenograft rejection are major obstacles in achieving long-term graft survival. Regulatory T cells have been proven to play a very important role in the regulation of immune responses to self or non-self Ags. Previous studies have shown that TCRαβ+CD3+CD4−CD8− (double-negative (DN)) T cells possess an immune regulatory function, capable of controlling antidonor T cell responses in allo- and xenotransplantation through Fas-Fas ligand interaction. In this study, we investigated the possibility that xenoreactive DNT cells suppress B cells. We found that DNT cells generated from wild-type C57BL/6 mice expressed B220 and CD25 after rat Ag stimulation. These xenoreactive B220+CD25+ DNT cells lysed activated, but not naive, B and T cells. This killing, which took place through cell-cell contact, required participation of adhesion molecules. Our results indicate that Fas ligand, TGF-β, TNF-α, and TCR-MHC recognition was not involved in DNT cell-mediated syngenic cell killing, but instead this killing was mediated by perforin and granzymes. The xenoreactive DNT cells expressed high levels of granzymes in comparison to allo- or xenoreactive CD8+ T cells. Adoptive transfer of DNT cells in combination with early immune suppression by immunosuppressive analog of 15-deoxyspergualin, LF15-0195, significantly prolonged rat heart graft survival to 62.1 ± 13.9 days in mice recipients. In conclusion, this study suggests that xenoreactive DNT cells can control B and T cell responses in perforin/granzyme-dependent mechanisms. DNT cells may be valuable in controlling B and T cell responses in xenotransplantation.

Synergistic tolerance induced by LF15-0195 and anti-CD45RB monoclonal antibody through suppressive dendritic cells.

Background. LF 15-0195 (LF), a novel analogue of 15-deoxyspergualin (DSG), inhibits maturation of dendritic cells (DC). Anti-CD45RB is a monoclonal antibody (mAb) that blocks activation of T-helper (Th) 1 cells and generates T-regulatory cells. This study addressed whether these two reagents act synergistically to inducing tolerance, and investigated associated cellular mechanisms. Methods. BALB/c recipients were treated by a short course of mAb alone, LF alone, or the combination of both agents. Mice that accepted a C57BL/6 cardiac allograft for more than 100 days were considered tolerant. Splenic DC were purified using positive selection for CD11c. Bone marrow DC were generated by culture with interleukin-4 and granulocyte-macrophage colony-stimulating factor. Surface marker expression was determined by fluorescence-activated cell sorter analysis. DC function was assessed by the ability to stimulate or inhibit T cells in vitro. Results. Although monotherapy with LF or mAb failed to induce tolerance, combination therapy resulted in long-lasting acceptance of allogeneic hearts (>200 days) and secondary donor skin grafts (>100 days). DC from tolerant recipients possessed lower major histocompatibility complex class II and CD40 expression, and were poorer co-stimulators for T-cell proliferation than control DC. Furthermore, DC from tolerant mice induced Th2 differentiation, suppressed overall T-cell proliferation, and were poor presenters of T cells specific for antigen to pigeon cytochrome c 81–104. Conclusions. The combination of LF and anti-CD45RB mAb induced stable tolerance. The synergy of these two approaches appears to be mediated through formation of tolerogenic DC and T-regulatory cells.

Natural Killer Cells Mediate Long-term Kidney Allograft Injury.

Background Chronic allograft injury remains the leading cause of late kidney graft loss despite improvements in immunosuppressive drugs and a reduction in acute T cell–mediated rejection. We have recently demonstrated that natural killer (NK) cells are cytotoxic to tubular epithelial cells and contribute to acute kidney ischemia-reperfusion injury. The role of NK cells in kidney allograft rejection has not been studied. Methods A “parent to F1” kidney transplant model was used to study NK cell–mediated transplant rejection. Results The C57BL/6 kidneys were transplanted into fully nephrectomized CB6F1 (C57BL/6 x BALB/c) mice. Serum creatinine levels increased from baseline (18.8 ± 5.0 &mgr;mol/L to 37.2 ± 5.9 &mgr;mol/L, P < 0.001) at 60 days after transplantation. B6Rag−/−-to-CB6F1Rag−/− (B6Rag−/−xBALB/cRag−/−) recipients, which lack T and B cells but retain NK cells, showed similar levels of kidney dysfunction 65 days after transplantation (creatinine, 33.8 ± 7.9 &mgr;mol/L vs 17.5 ± 5.1 &mgr;mol/L in nontransplant Rag−/− mice, P < 0.05). Importantly, depletion of NK cells in Rag1−/− recipients inhibited kidney injury (24.6 ± 5.5 &mgr;mol/L, P < 0.05). Osteopontin, which can activate NK cells to mediate tubular epithelial cell death in vitro, was highly expressed in 60 days kidney grafts. Osteopontin null kidney grafts had reduced injury after transplantation into CB6F1 mice (17.7 ± 3.1 &mgr;mol/L, P < 0.001). Conclusions Collectively, these data demonstrate for the first time that independent of T and B cells, NK cells have a critical role in mediating long-term transplant kidney injury. Specific therapeutic strategies that target NK cells in addition to conventional immunosuppression may be required to attenuate chronic kidney transplant injury.

Monotherapy with LF 15-0195, an analogue of 15-deoxyspergualin, significantly prolongs renal allograft survival in monkeys

Background. LF 15-0195 is a novel, more potent, and less toxic analogue of 15-deoxyspergualin, an antibiotic used as an immunosuppressive agent to prevent rejection of organ transplants. This study was undertaken to determine whether LF 15-0195 monotherapy would prevent renal allograft rejection in a nonhuman primate model. Methods. In the study groups, recipients received LF 15-0195 monotherapy at doses of 0.065 mg/kg per day (group 2, n=4), 0.13 mg/kg per day (group 3, n=4), or 0.2 mg/kg per day (group 4, n=4), administered subcutaneously, on postoperative days 0 to 14. Results. Group 1 consisted of untreated control recipients, all of which developed advanced graft rejection after surviving for an average of 6.5±0.6 days. LF 15-0195 treatment significantly prolonged graft survival in groups 2, 3, and 4, to 20±20 days, 49±5 days, and 39±4 days, respectively. Animals in groups 3 and 4 demonstrated no evidence of rejection during LF 15-0195 treatments. The animals maintained stable renal function for 2 weeks after LF 15-0195 withdrawal but gradually developed rejection at 5 to 6 weeks. Pathologic studies demonstrated that vascular graft rejection was attenuated in LF 15-0195-treated allografts, compared with control specimens. These groups also demonstrated transient reductions in lymphocyte counts during treatment, which returned to normal levels 2 weeks after LF 15-0195 withdrawal. Total serum concentrations of IgM and IgG decreased by a mean of 20.4% and a mean of 31.4%, respectively, at the end of LF 15-0195 treatment (postoperative day 14). LF 15-0195 did not significantly alter thrombocyte counts or hemoglobin levels. Necropsy studies showed no evidence of drug toxicity in the heart, liver, spleen, intestines, stomach, or colon. Conclusions. LF 15-0195 monotherapy significantly prolonged renal allograft survival in monkeys. These encouraging data suggest that this novel agent may be of future value in clinical transplantation.

Natural killer cells play a critical role in cardiac allograft vasculopathy in an interleukin-6--dependent manner.

Background Approximately 50% of cardiac transplants fail in the long term, and currently, there are no specific treatments to prevent chronic rejection. In the clinic, donor cardiac graft ischemia time is limited to within a few hours and correlates with delayed graft function and organ failure. It is still unknown how ischemic injury negatively influences allograft function over the long term despite advances in immunosuppression therapy. Methods Allogeneic cardiac grafts were stored at 4°C for 4 hr before being transplanted into T/B cell–deficient Rag−/− mice or T/B/natural killer (NK) cell–deficient &ggr;c−/−Rag−/− mice. Grafts were harvested 60 days after transplantation and indicators of chronic allograft vasculopathy (CAV) were quantified. Results We have found that cold ischemia of cardiac grafts induces CAV after transplantation into Rag1−/− mice. Interestingly, cold ischemia–induced CAV posttransplantation was not seen in T/B/NK cell–deficient &ggr;c−/−Rag−/− mice. However, cardiac grafts in &ggr;c−/−Rag−/− mice that received an adoptive transfer of NK cells developed CAV, supporting the role of NK cells in CAV development. Analysis of various cytokines that contribute to NK cell function revealed high interleukin (IL)-6 expression in cardiac grafts with CAV. In addition, IL-6–deficient cardiac grafts did not develop CAV after transplantation into allogeneic Rag−/− mice. Conclusion These data demonstrate that cold ischemia and NK cells play critical roles in the development CAV. Natural killer cells and injured grafts may play a reciprocal role for CAV development in an IL-6–independent manner. Specific therapeutic strategies may be required to attenuate NK cell contribution to chronic cardiac rejection.

Distinct Subsets of Dendritic Cells Regulate the Pattern of Acute Xenograft Rejection and Susceptibility to Cyclosporine Therapy

We determined whether distinct subclasses of dendritic cells (DC) could polarize cytokine production and regulate the pattern of xenograft rejection. C57BL/6 recipients, transplanted with Lewis rat hearts, exhibited a predominantly CD11c+CD8α+ splenic DC population and an intragraft cytokine profile characteristic of a Th1-dominant response. In contrast, BALB/c recipients of Lewis rat heart xenografts displayed a predominantly CD11c+CD8α− splenic DC population and IL-4 intragraft expression characteristic of a Th2 response. In addition, the CD11c+IL-12+ splenic DC population in C57BL/6 recipients was significantly higher than that in BALB/c recipients. Adoptive transfer of syngeneic CD8α− bone marrow-derived DC shifted a Th1-dominant, slow cell-mediated rejection to a Th2-dominant, aggressive acute vascular rejection (AVR) in C57BL/6 mice. This was associated with a cytokine shift from Th1 to Th2 in these mice. In contrast, transfer of CD8α+ bone marrow-derived DC shifted AVR to cell-mediated rejection in BALB/c mice and significantly prolonged graft survival time from 6.0 ± 0.6 days to 14.2 ± 0.8 days. CD8α+ DC transfer rendered BALB/c mice susceptible to cyclosporine therapy, thereby facilitating long-term graft survival. Furthermore, CD8α+ DC transfer in IL-12-deficient mice reconstituted IL-12 expression, induced Th1 response, and attenuated AVR. Our data suggest that the pattern of acute xenogeneic rejection can be regulated by distinct DC subsets.