Donor Alloantigens Research Articles

Tolerogenic Dendritic Cell-Regulatory T-cell Interaction and the Promotion of Transplant Tolerance

Evidence has accumulated that targeting of donor alloantigen to quiescent dendritic cells (DC) in situ or adoptive DC therapy is associated with the expansion or induction of regulatory T cells (Treg) in experimental organ transplantation. These Treg can mediate suppression of antidonor T-effector cell responses and promote allograft tolerance. In addition, Treg can exert reciprocal, inhibitory effects on DC that maintain their tolerogenic properties. Several groups have exploited DC to expand allo-Ag-specific Treg in vitro, followed by adoptive transfer of the Treg to graft recipients, an approach that holds promise for tolerogenic cell therapy in clinical cell and organ transplantation.

Dendritic cell (DC)- derived exosomes spread donor allo-antigen between recipient's DC following cardiac transplantation (141.11)

Abstract Exosomes are nanovesicles generated in multivesicular endosomes that are released to the extracellular space by different cell types. Since DC-derived exosomes (dexosomes) express MHC-peptide and costimulatory molecules, we investigated the role of dexosomes in elicitation of the anti-donor response in transplantation. Dexosomes were generated from bone marrow-derived DC and labeled with PKH67 for traffic studies. CD4+ TCRtg T cells (Thy1.1+) specific for the IEα52-68 (BALB/c)-IAb (B10) complex were CFSE-labeled and transferred i.v. to host Thy1.2+ B6 mice. Graft infiltrating DC were isolated from BALB/c (CD45.2+) cardiac grafts 3 days after transplantation in B6 (CD45.2+) recipients and genetically-engineered ex vivo to release exosomes expressing the reporter marker eGFP, and then transferred i.v. into host CD45.1+ B6 mice. We demonstrated that although graft-infiltrating leukocytes release exosomes ex vivo, they do not secrete enough exosomes in circulation to stimulate donor-reactive T-cells in lymphoid organs. Instead, migrating DCs (generated in vitro or isolated from allografts), once they home in the spleen, they transfer exosomes expressing eGFP to spleen-resident DCs. Thus, exchange of exosomes between DCs in lymphoid organs constitutes a mechanism by which passenger leukocytes transfer alloAg to recipient's APC and amplify generation of donor-reactive T-cells.

Immunoprotection by polyethylene glycol in organ preservation solutions is not due to an immunomasking effect

New organ preservation solutions that contain soluble polyethylene glycol (sPEG) molecules have been associated with reduction of acute rejection episodes. In the present manuscript we tested in vitro whether sPEG molecules were able to mask donor alloantigens and reduce graft immunogenicity. Immunomasking effect was only evidenced when PEG molecules were covalently bound to donor cell surface. We concluded that sPEG in preservation solution are unlikely to display 'immunocamouflage' property.

Combined therapy of CD4 +CD25 + regulatory T cells with low-dose sirolimus, but not calcineurin inhibitors, preserves suppressive function of regulatory T cells and prolongs allograft survival in mice

Our previous study proved that sirolimus is a potent immunosuppressant which induces long-term allograft survival depends on persistence of alloantigens. CD4 +CD25 + regulatory T (Treg) cells are potent suppressors in transplantation. Our objectives focus on whether combined-therapy of Tregs with immunosuppressants could prolong allograft survival in mice. The study showed that inhibition of Tregs was maintained by co-cultured with sirolimus (1 nM) in vitro, but not tacrolimus (1 nM) or CsA (1 nM). When the concentration was increased > 100 nM, suppression was fallen. Based on the ability of sirolimus to target effector T cells, but retaining the inhibition of Tregs, an adoptive infusion of donor alloantigen specific Tregs combined with 30-day sirolimus (1 mg/kg) and 3-day ATG (20 mg/kg) was found to prolong heart allograft survival in mice. Even though the cell numbers of CD4 + T cells were found to decrease in sirolimus-treated mice, sirolimus selectively enhanced the numbers of CD4 +CD25 + cells and increased the expression of Foxp3 in spleens and lymph nodes, respectively, in recipients. However, combined therapy with low-dose CsA (5 mg/kg) or tacrolimus (1 mg/kg) reduced significantly the expression of Foxp3 and failed to prolong the allograft survival. In summary, expanded Tregs exposed to sirolimus can survive, proliferate, and preserve inhibition in vitro. Tregs are more resistant to sirolimus than other T cells. Combined with Tregs, sirolimus rather than calcineurin inhibitors, prolongs the allograft survival. Sirolimus may be the best copartner for Tregs therapy. It also suggests calcineurin-dependent signals may be required in the development of Tregs.

Alloantigen specific T regulatory cells in transplant tolerance

CD4 +CD25 +Foxp3 +T cells are regulatory/suppressor cells (Treg) that include non-antigen(Ag)-specific as well as Ag-specific Tregs. How non-Ag-specific naïve CD4 +CD25 +Treg develop into specific Tregs is unknown. We have studied DA rats tolerant to fully allogeneic PVG cardiac grafts that survived with out immunosuppression for over 100 days and identified the cellular basis of alloantigen specific tolerance. Key observations from our studies will be reviewed including how CD4 +CD25 +Tregs were first identified and the cytokine dependence of CD4 +T cells that transfer alloantigen specific transplant tolerance which died in culture unless stimulated with both cytokine rich ConA supernatant and specific donor alloantigen. Both the tolerant CD4 +CD25 + and CD4 +CD25 − T cell populations are required to transfer tolerance, yet alone the CD4 +CD25 − T cell effect rejection. Tolerance transfer occurs with a low ratio of CD4 +CD25 +T cells (< 1:10), whereas to induce tolerance with naive CD4 +CD25 +T cells requires both a ratio of > 1:1 and is not alloantigen specific. Recent findings on how naïve CD4 +CD25 +T cells developed into two separated pathways of alloantigen specific Tregs, by culturing them with alloAg with either IL-2 or IL-4 and donor alloantigen are described. IL-2 enhances IFN-γR and IL-5 mRNA while IL-4 induced a reciprocal profile with de novo IL-5Rα and increased IFN-γ mRNA expression. Both IL-2 and IL-4 alloactivated CD4 +CD25 +Tregs within 3–4 days of culture can induce alloantigen specific tolerance at ratios of 1:10. Long term, CD4 +CD25 +T cells from tolerant hosts given IL-2 cultured cells have increased IL-5 and IFN-γR mRNA; whereas hosts given IL-4 cultured cells had enhanced IL-5Rα mRNA expression and IL-5 enhanced their proliferation to donor but not third party alloAg. These findings suggest that Th1 and Th2 responses activate two pathways of alloantigen specific Tregs that can mediate transplant tolerance but are dependent upon cytokines produced by ongoing Th1 and/or Th2 immune responses.

Donor-Specific Tolerance Induction through Expansion of DCregs and Tregs by a Liposomal Formulation of KRN7000 (RGI-2001).

Pharmacological induction of donor-specific tolerance would provide significant benefits in both organ transplantation and bone marrow transplantation settings. We investigated the ability of alpha-galactosylceramide (α-GC) in inducing donor-specific tolerance when given in a liposomal formulation. α-GC is a ligand for CD1d molecules expressed on antigen-presenting cells. Upon presentation by CD1d to invariant natural killer T (iNKT) cells, α-GC induces the rapid release of Th1, Th2, or immune regulatory cytokines and initiation of multiple downstream cellular events such as T cell polarization and expansion of dendritic cell subsets. Previously, using RGI-2001 (a liposomal formulation of a synthetic derivative of α-GC, KRN7000), we have demonstrated that the activation of iNKT cells by RGI-2001 induces expansion of regulatory dendritic cells (DCreg) and subsequent generation of antigen-specific Foxp3+ regulatory T (Treg) cells in the presence of target antigens. In the present study, we examined the effects of RGI-2001 on immune responses against alloantigens using a murine in vivo experimental system. In brief, Balb/c (H-2d) recipients were primed with 5x10e6 C57BL/6 (H-2b) whole spleen cells (WSC) with varying doses of RGI-2001 (0.002 to 20 μg/mouse) given intravenously. Seven days later, WSC from the Balb/c recipients were examined for their cellular composition by FACS analysis. Subtle but reproducible dose-dependent increases were noted in the percentage of the LinnegCD11cintCD45RB+ dendritic cell (DC) population, known to be enriched for regulatory DC (DCreg). Since RGI-2001 induces an increase in the total spleen cells, the absolute DCreg cell numbers in RGI-2001-treated spleen increased in a statistically significant manner as compared with untreated controls. As for the percentages of CD4+Foxp3+ Treg cells, no apparent differences were observed. However, an analysis using the Ki67 cycling cell specific nuclear marker revealed a clear dose-dependent increase in the cycling cell fraction among CD4+Foxp3+ Treg cells. These results together confirmed that RGI-2001 induces expansion of DCregs and Tregs. Next, we investigated the effects of RGI-2001 on immune responses against the donor alloantigens. The WSC of the recipient mice were restimulated in vitro with the mitomycin C-treated, T cell depleted donor (C57BL/6) WSC, and the levels of proliferation were measured by MTT colorimetric assay (one-way MLR). It was found that RGI-2001 treatment reproducibly and significantly suppressed proliferation of host WSC in response to donor alloantigens. A dose of 2μg/mouse of RGI-2001 induced in average ~30% reduction in host WSC proliferation. IL-2 production was also reduced to ~50%, further indicating the suppressive effects of RGI-2001. Notably, it was confirmed that suppressive effect of RGI- 2001 was restricted to the responses towards donor specific alloantigens, as no suppression in proliferation nor IL-2 production was noted when a third party (C3H) WSC was used as the stimulators. Collectively, the results suggest that RGI-2001, when administered together with allogeneic donor cells, can induce donor specific tolerance by expanding DCregs and inducing antigen-specific Tregs. RGI-2001 may have a potential to be a novel therapy to prevent organ rejection as well as GvHD in bone marrow transplantation.

Sensitization to Minor Histocompatibility Antigens Poses a Barrier in Transplantation and Can Be Prevented by CD154:CD40 Co-Stimulatory Blockade.

There is an increased rate of graft failure associated with nonmyeloablative conditioning approaches for hematopoietic stem cell (HSC) transplantation, often resulting in subsequent sensitization to donor alloantigens. Recipient sensitization is among the most critical of problems currently facing clinical transplantation, resulting in hyperacute rejection of transplanted HSC and/or organ grafts by existing donor-specific antibodies (Ab) against major histocompatibility (MHC) antigens in secondary transplants. The humoral immune response to minor histocompatibility antigens (Mi-HAg) has not been fully evaluated in BMC transplantation to date. In this study, we explored the role of allosensitization to Mi-HAg in BMC transplant rejection using a mouse model. AKR (H-2k) mice were sensitized with donor skin grafts from MHC-matched, but minor antigen-disparate B10.BR (H-2k) mice. Significantly higher levels of Abs against donor were generated as detected at 4 weeks after skin grafting by flow cytometry cross match assay (mean fluorescence intensity (MFI) 231.3 ± 151.1; P < 0.0001) compared with the Ab levels in sera of naïve AKR mice (MFI 4.2 ± 2.0) . The MFI of Ab titer to anti-Mi-HAg was similar to that in mice after rejecting skin grafts with MHC disparity (MFI: 231.3 ± 139.0) or MHC plus Mi-HAg disparity (MFI: 301.1 ± 141.1). IgG subclasses of IgG1, IgG2a, and IgG2b were detected in all tested AKR mice with high levels of anti–donor total IgG. Surprisingly, the Abs generated to minor antigens were not donor-specific as they also bound to splenocytes from B6 (H-2b), BALB/c (H-2d), C3H (H-2k) and B10.D2 (H-2d) mice. In subsequent HSC transplantation performed 5–7 weeks after skin grafting, only 20% of B10.BR skin sensitized AKR mice engrafted after ablative conditioning. In contrast, all naïve AKR control mice engrafted but none did in mice sensitized to MHC alloantigens. These findings correlated with in vivo cytotoxicity assays performed at the same time point. Mi-HAg sensitized AKR mice eliminated CFSE labeled donor splenocytes with a significantly faster rate than naïve AKR but significantly slower rate than the mice sensitized to MHC alloantigen. We previously reported that blockade of signaling via the CD40/CD154 co-stimulatory pathway with anti-CD154 induces B cell tolerance and abrogates donor-specific Ab generation in a MHC plus minor antigen mismatched mouse model. Here, we therefore examined whether CD154 blockade during exposure to minor antigens would also prevent sensitization to minor antigens. The production of anti-donor-Mi-HAg antibodies was totally prevented in mice treated with anti-CD154 mAb (MFI 6.1±3.48l P=0.33) compared with the Ab in naïve mice (3.9±0.25), suggesting a dominant role of the CD154:CD40 pathway in B-cell responses to Mi-HAg. Moreover, anti-CD154 treatment promoted bone marrow engraftment to 100% in recipients previously exposed to donor Mi-HAg, suggesting that antibody and not T cells play a dominant role in sensitization to minor antigens. These data demonstrate that sensitization to minor antigens poses a significant barrier in transplantation, but with less intensity than sensitization to MHC antigens, and can be prevented by anti-CD154 mAb. Our findings may identify a novel and clinically relevant approach in the development of conditioning approaches to enhance engraftment but prevent sensitization in the event of graft failure.

Treatment and Prophylaxis of Cardiac Allograft Vasculopathy

Cardiac allograft vasculopathy (CAV) remains a life-threatening complication after heart transplantation (HT). Recipients with severe intimal thickening are 10-fold more likely to suffer cardiac events than those without severe hyperplasia. From July 1987 to July 2007, we performed 323 HTs with 5-year actuarial freedom from CAV of 69%, similar to the data reported by the International Society for Heart and Lung Transplantation, namely, 68% at 5 years. Therefore, CAV is not uncommon in Asia. The pathogenesis of CAV is initial endothelial injury followed by intimal hyperplasia and proliferation of vascular smooth muscle cells. It may be caused by both immunological events (involving T or B cells in response to donor major histocompatibility antigens, or natural killer [NK] cell-triggered recruitment of T cells not responsive to donor alloantigen) and nonimmunologic factors, such as older age, ischemia-reperfusion injury, viral infection (particularly cytomegalovirus [CMV] infection), immunosuppressive drugs, and classic risk factors, such as hyperlipidemia, insulin resistance, and hypertension. The therapy for CAV has been disappointing, despite prescriptions of statin lipid-lowering agents, calcium-channel blockers, angiotensin-converting enzyme inhibitors, and antiproliferative drugs. Patients with CAV are often not amenable to successful revascularization (medical or surgical) because of the diffuse obliterative process. Prophylaxis of CAV starts with modification of risk factors: hypertension, hyperlipemia, hyperglycemia, obesity, and smoking, as well as promotion of exercise programs. The HMG-CoA reductase inhibitors and antiproliferative drugs may slow the progression of CAV by various immunologic and nonimmunologic effects. Prevention of CMV infection reduces CAV. Mycophenolate mofetil and signal transduction inhibitors, such as everolimus, reduce intimal thickening and CAV.

The Classical Complement Pathway in Transplantation: Unanticipated Protective Effects of C1q and Role in Inductive Antibody Therapy

Though complement (C) deposition within the transplant is associated with allograft rejection, the pathways employed have not been established. In addition, evidence suggests that C-mediated cytolysis may be necessary for the tolerance-inducing activities of mAb therapies. Hence, we assessed the role of the classical C pathway in acute allograft rejection and its requirement for experimental mAb therapies. C1q-deficient (C1q-/-) recipients rejected allografts at a faster rate than wild-type (WT) recipients. This rejection was associated with exacerbated graft pathology but not with enhanced T-cell responses in C1q-/- recipients. However, the humoral response to donor alloantigens was accelerated in C1q-/- mice, as an early IgG response and IgG deposition within the graft were observed. Furthermore, deposition of C3d, but not C4d was observed in grafts isolated from C1q-/- recipients. To assess the role of the classical C pathway in inductive mAb therapies, C1q-/- recipients were treated with anti-CD4 or anti-CD40L mAb. The protective effects of anti-CD4 mAb were reduced in C1q-/- recipients, however, this effect did not correlate with ineffective depletion of CD4+ cells. In contrast, the protective effects of anti-CD40L mAb were less compromised in C1q-/- recipients. Hence, this study reveals unanticipated roles for C1q in the rejection process.

A cell-based approach to the minimization of immunosuppression in renal transplantation

Five renal transplant recipients were preoperatively treated with transplant acceptance-inducing cells (TAICs) in a Phase-I safety study of TAICs as an adjunct immune-conditioning therapy in living-donor kidney transplantation. Initially, patients received anti-thymocyte globulin induction therapy in combination with tacrolimus and steroid immunosuppression. Over the course of 12 weeks, steroids were withdrawn and tacrolimus therapy was minimized. Three of the five patients were able to tolerate low-dose tacrolimus monotherapy and one patient was withdrawn from all immunosuppression for over 8 months. No acute or delayed adverse events were associated with the infusion of TAICs. Monitoring of the recipient anti-donor reactivity of TAIC-treated patients in mixed lymphocyte cultures demonstrated that, during periods of clinically stable graft function, recipient T-cell proliferation and cytokine secretion in response to stimulation with donor alloantigen was relatively suppressed. Therefore, although the TAIC-II trial did not provide conclusive evidence of a beneficial effect of preoperative TAIC treatment, the results were encouraging because they suggest that TAICs promote a state of alloantigen-specific unresponsiveness, which might allow safe minimization of pharmacological immunosuppression.

Alloantigen-Pulsed Host Dendritic Cells Induce T-Cell Regulation and Prolong Allograft Survival in a Rat Model of Hindlimb Allotransplantation

Composite tissue allotransplantation is restricted due to the risks presented by long-term therapeutic immunosuppression. This study is conducted to investigate whether treatment with recipient immature dendritic cells (DCs) pulsed with donor alloantigens can prolong allograft survival and induce T-cell regulation in a rodent model. Orthotopic hindlimb transplants from Brown-Norway (RT1(n)) to Lewis (RT1(1)) rats were performed (day 0). DCs were propagated from the recipient bone marrow and pulsed with the donor alloantigen lysate. Group 1 (control group) did not receive any treatment. Groups 2 and 3 received cyclosporine A (CsA) at a concentration of 10 and 16 mg.kg(-1).day(-1), respectively, on days 0-20 following composite tissue allotransplantation. Group 4 received antilymphocyte serum (i.p. administered 4 d before and 1 d after transplantation) therapy. Group 5 received combined treatment with CsA (10 mg.kg(-1).day(-1), days 0-20) and donor alloantigen-pulsed recipient DCs (i.v. administered on days 7, 14, and 21). Group 6 received combined treatment with CsA (10 mg.kg(-1).day(-1) on days 0-20), antilymphocyte serum (administered i.p. 4 d before and 1 d after transplantation), and DCs (administered i.v. on days 7, 14, and 21). Graft rejection was defined as epidermolysis/desquamation of the donor skin. The mixed lymphocyte reaction was performed to determine the donor T-cell reactivity. Tissue samples were biopsied to analyze the histological changes, and flow cytometry was performed to quantify the donor T-cells. Allograft survival was significantly prolonged (>200 d) in Group 6 when compared with the other groups (P < 0.001). The mixed lymphocyte reaction performed for Group 6 revealed hyporesponsiveness of the T-cells to donor alloantigens. Flow cytometric analysis in Group 6 revealed a significant increase in the percentage of CD4(+)/CD25(+) and CD4(+)/foxP3(+) T-cells expression, and significant increase in the percentage of donor cells (RT1(n)) in the recipient peripheral blood. Immunohistochemical staining of allo-skin revealed a significant increase in the proportion of CD25(+) cells in the subcutaneous and dermis layers in Group 6, as compared to other groups. Treatment with donor alloantigen-pulsed recipient immature DCs in combination with transient immunosuppression prolongs allograft survival and induced tolerance by inducing T-cell hyporesponsiveness to donor alloantigens and increasing the CD4(+)/CD25(+) T-cell population.

Alloreactive CD8 T Cell Tolerance Requires Recipient B Cells, Dendritic Cells, and MHC Class II

Allogeneic bone marrow chimerism induces robust systemic tolerance to donor alloantigens. Achievement of chimerism requires avoidance of marrow rejection by pre-existing CD4 and CD8 T cells, either of which can reject fully MHC-mismatched marrow. Both barriers are overcome with a minimal regimen involving anti-CD154 and low dose (3 Gy) total body irradiation, allowing achievement of mixed chimerism and tolerance in mice. CD4 cells are required to prevent marrow rejection by CD8 cells via a novel pathway, wherein recipient CD4 cells interacting with recipient class II MHC tolerize directly alloreactive CD8 cells. We demonstrate a critical role for recipient MHC class II, B cells, and dendritic cells in a pathway culminating in deletional tolerance of peripheral alloreactive CD8 cells.

Exhaustive Differentiation of Alloreactive CD8+ T Cells: Critical for Determination of Graft Acceptance or Rejection

The precise role that CD8+ T cells play in the rejection and acceptance of different types of allograft is unclear and has been shown to vary between donor-recipient combinations. The response of adoptively transferred CD8+ T cells reactive to the donor alloantigen H2Kb was examined after transplantation of H2Kb liver, kidney, and heart grafts in mice. After transfer of 6 x 10(6) alloreactive CD8+ T cells to T-cell depleted syngeneic mice spontaneous long-term acceptance of liver grafts was observed, whereas kidney and heart grafts were acutely rejected. Within 5 days of liver transplantation, we found that the entire H2Kb-reactive T-cell pool was stimulated to proliferate and differentiate into memory or effector cells that were detectable within lymphoid tissues as well as the liver graft itself. However, despite the generation of effector or memory T cells, liver allografts were accepted, which correlated with the exhaustion or deletion of such cells. In contrast, although activation and proliferation of H2Kb-reactive CD8+ T cells was observed after transplantation of heart or kidney grafts, unactivated, H2Kb-reactive CD8+ T cells were still present in the spleen even long term. Interestingly, differences in the effector function of liver and kidney graft infiltrating donor-reactive CD8+ T cells were not detected after adoptive transfer into immunodeficient mice, despite a reduction in Th1-type cytokines within liver grafts. The rapid and extensive initial activation and differentiation of donor-reactive CD8+ T cells that occurs after liver transplantation leads to clonal exhaustion or deletion of the alloreactive CD8+ T-cell repertoire resulting in spontaneous tolerance induction.

Development of Either Split Tolerance or Robust Tolerance along with Humoral Tolerance to Donor and Third-Party Alloantigens in Nonmyeloablative Mixed Chimeras

Hematopoietic chimerism is considered to generate robust allogeneic tolerance; however, tissue rejection by chimeras can occur. This "split tolerance" can result from immunity toward tissue-specific Ags not expressed by hematopoietic cells. Known to occur in chimeric recipients of skin grafts, it has not often been reported for other donor tissues. Because chimerism is viewed as a potential approach to induce islet transplantation tolerance, we generated mixed bone marrow chimerism in the tolerance-resistant NOD mouse and tested for split tolerance. An unusual multilevel split tolerance developed in NOD chimeras, but not chimeric B6 controls. NOD chimeras demonstrated persistent T cell chimerism but rejected other donor hematopoietic cells, including B cells. NOD chimeras also showed partial donor alloreactivity. Furthermore, NOD chimeras were split tolerant to donor skin transplants and even donor islet transplants, unlike control B6 chimeras. Surprisingly, islet rejection was not a result of autoimmunity, since NOD chimeras did not reject syngeneic islets. Split tolerance was linked to non-MHC genes of the NOD genetic background and was manifested recessively in F(1) studies. Also, NOD chimeras but not B6 chimeras could generate serum alloantibodies, although at greatly reduced levels compared with nonchimeric controls. Surprisingly, the alloantibody response was sufficiently cross-reactive that chimerism-induced humoral tolerance extended to third-party cells. These data identify split tolerance, generated by a tolerance-resistant genetic background, as an important new limitation to the chimerism approach. In contrast, the possibility of humoral tolerance to multiple donors is potentially beneficial.

Costimulatory blockade of CD154-CD40 in combination with T-cell lymphodepletion results in prevention of allogeneic sensitization

AbstractSensitization is a critical unresolved challenge in transplantation. We show for the first time that blockade of CD154 alone or combined with T-cell depletion prevents sensitization. Allogeneic skin grafts were rejected by recipients treated with anti-αβ T-cell receptor (TCR), anti-CD154, anti-OX40L, or anti–inducible costimulatory pathway (ICOS) mAb alone with a kinetic similar to untreated recipients. However, the production of anti–donor MHC antibody was prevented in mice treated with anti-CD154 mAb only, suggesting a specific role for the CD154-CD40 pathway in B-cell activation. The impairment of T cell–dependent B-cell responses by blocking CD154 occurs through inhibiting activation of T and B cells and secretion of IFN-γ and IL-10. Combined treatment with both anti-CD154 and anti–αβ TCR abrogated antidonor antibody production and resulted in prolonged skin graft survival, suggesting the induction of both T- and B-cell tolerance with prevention of allogeneic sensitization. In addition, we show that the tolerance induced by combined treatment was nondeletional. Moreover, these sensitization-preventive strategies promote bone marrow engraftment in recipients previously exposed to donor alloantigen. These findings may be clinically relevant to prevent allosensitization with minimal toxicity and point to humoral immunity as playing a dominant role in alloreactivity in sensitized recipients.

Induction of transplantation tolerance by combining non-myeloablative conditioning with delivery of alloantigen by T cells

The observation that bone marrow derived hematopoietic cells are potent inducers of tolerance has generated interest in trying to establish transplantation tolerance by inducing a state of hematopoietic chimerism through allogeneic bone marrow transplantation. However, this approach is associated with serious complications that limit its utility for tolerance induction. Here we describe the development of a novel approach that allows for tolerance induction without the need for an allogeneic bone marrow transplant by combining non-myeloablative host conditioning with delivery of donor alloantigen by adoptively transferred T cells. CBA/Ca mice were administered 2.5 Gy whole body irradiation (WBI). The following day the mice received K b disparate T cells from MHC class I transgenic CBK donor mice, as well as rapamycin on days 0–13 and anti-CD40L monoclonal antibody on days 0–5, 8, 11 and 14 relative to T cell transfer. Mice treated using this approach were rendered specifically tolerant to CBK skin allografts through a mechanism involving central and peripheral deletion of alloreactive T cells. These data suggest robust tolerance can be established without the need for bone marrow transplantation using clinically relevant non-myeloablative conditioning combined with antigen delivery by T cells.

Ex Vivo-Expanded Natural CD4+CD25+ Regulatory T Cells Synergize With Host T-Cell Depletion to Promote Long-Term Survival of Allografts

Foxp3(+)CD4(+)CD25(+) natural regulatory T (nT(reg)) cells have been shown in immunodeficient mice to suppress allograft rejection after adoptive cotransfer. We hypothesized that immunotherapy using ex vivo-expanded nT(reg) could suppress allograft rejection in wild-type mice. Donor alloantigen (alloAg) specificity of naive splenic nT(reg) was enriched in vitro by culturing with anti-CD3/CD28-coated Dynabeads plus bone marrow-derived dendritic cells (BM-DC) in the presence of interleukin (IL)-2 or IL-2 plus transforming growth factor (TGF)-beta. On average, 96.2% fresh CD4(+)CD25(+) nT(reg) were intracellular Foxp3(+). By d+20 in culture, 6.4% nT(reg) were Foxp3(+) following expansion with IL-2 alone, and 14.4% or 19.7% nT(reg) were Foxp3(+) when expanded with IL-2 plus 0.5 or 2.5 ng/mL TGF-beta, respectively. In vitro, alloAg-enriched, TGF-beta/IL-2-conditioned nT(reg) exerted stronger donor alloAg-specific suppression than cells with IL-2 alone in mixed lymphocyte reaction (MLR) assays. In vivo, alloAg-enriched, TGF-beta/IL-2-conditioned nT(reg) expressed high-level Foxp3 following infusion, effectively overcame acute rejection and induced long-term survival of donor but not third-party heart allografts in peritransplant host T-cell-depleted mice. Long-term surviving allografts were noted to possess Foxp3(+) graft-infiltrating cells of exogenous and endogenous origins. In conjunction with transient host T-cell depletion, therapeutic use of ex vivo-expanded nT(reg) may be a practical means of preventing acute allograft rejection.

Perioperative donor splenocyte infusion induces regulatory T cells and abolishes acute pulmonary allograft rejection in miniature swine

Purpose: Donor alloantigen infusion has been shown to induce T cell regulation and transplant tolerance in small animal models. Here, we wished to study the effect of a donor splenocyte infusion on long term pulmonary allograft survival in a large animal model.

Alloantigen-specific prolongation of allograft survival in recipient mice treated by alloantigen immunization following ultraviolet-B irradiation

It is well documented that ultraviolet (UV) radiation present in sunlight suppresses immune responses. However, the majority of studies documenting the immunosuppressive effects of UV irradiation have been carried out in animals exposed to UV irradiation before immunization. Here, we report that recipient mice exposed to UV irradiation 7 days after immunization with a donor alloantigen exhibited prolongation of allograft survival in an alloantigen-specific manner. Recipient mice (H-2 b) intravenously immunized with 2 × 10 7 allogeneic spleen cells (H-2 b/d) 7 days before UV irradiation (40 kJ/m 2) showed prolonged survival of allografts presenting the alloantigen used for sensitization (H-2 b/d), but not third-party allografts (H-2 b/k). Adoptive transfer experiments revealed that CD4 + T cells in UV-irradiated recipients were responsible for this prolongation. CD4 + T cells that could transfer the suppression produced large amounts of interleukin (IL)-10, but not IL-4. The effect of UV irradiation on alloantigen-specific immunosuppression was cancelled by administration of an anti-IL-10 monoclonal antibody. These results indicate that UV irradiation given after alloantigen immunization induces alloantigen-specific type 1 regulatory T cell-like regulatory T cells that prolong allograft survival and imply that the difficulties associated with predicting donor-related organ availability in transplantation can be dealt with, given the effectiveness of UV irradiation after immunization.

Blockade of the OX40 ligand prolongs corneal allograft survival

Although corneal transplantation is one of the most common tissue transplantations and is known to have a high graft acceptance rate, occasional corneal graft rejection remains a cause of blindness. OX40, a member of the TNF receptor superfamily, is expressed on activated T cells, and transmits a costimulatory signal by binding to OX40 ligand (OX40L) expressed on several cells with antigen-presenting functions. Using a blocking monoclonal antibody (mAb) against murine OX40L, we investigated the role of OX40 in a murine model of corneal transplantation. C3H/He mouse corneas were transplanted to BALB/c mice orthotopically. Administration of anti-OX40L mAb significantly reduced allograft rejection, and increased graft survival rate to 40% at 8 weeks after transplantation, while all corneas were rejected within 5 weeks in control IgG-treated mice. Similar reduced rejection was observed when wild-type donor corneas were transplanted to OX40L-deficient recipients. In vitro study revealed that the anti-OX40L mAb treatment reduced proliferative response and IFN-gamma production of draining lymph node cells in response to stimulation with donor alloantigen. These results demonstrate that OX40L blockade is effective for prolongation of corneal allograft survival by inhibiting recipient T cell activation.