Tolerance To Heart Allografts Research Articles

(494) - Donor Chimerism Duration Correlates with Heart Allograft Tolerance in Combined Heart and Bone Marrow Transplantation in Nonhuman Primates

(494) - Donor Chimerism Duration Correlates with Heart Allograft Tolerance in Combined Heart and Bone Marrow Transplantation in Nonhuman Primates

(91) - IL-6 Receptor Blockade Induces Tolerance of Heart Allografts in NHPs in a Simultaneous but Not Delayed Mixed-Chimerism Protocol

(91) - IL-6 Receptor Blockade Induces Tolerance of Heart Allografts in NHPs in a Simultaneous but Not Delayed Mixed-Chimerism Protocol

(360) Use of Anti-Il-6 or Anti-IL-6r Therapy in a Mixed Chimerism Protocol to Achieve Heart Allograft Tolerance in Non-Human Primates

(360) Use of Anti-Il-6 or Anti-IL-6r Therapy in a Mixed Chimerism Protocol to Achieve Heart Allograft Tolerance in Non-Human Primates

(357) Higher Frequency of Host Cd8+ Memory T Cells Correlates with Failure to Achieve Heart Allograft Tolerance via Mixed Chimerism and Il-6 Blockade in Multiparous Female Non-Human Primates

(357) Higher Frequency of Host Cd8+ Memory T Cells Correlates with Failure to Achieve Heart Allograft Tolerance via Mixed Chimerism and Il-6 Blockade in Multiparous Female Non-Human Primates

IL-2 receptor engineering enhances regulatory T cell function suppressed by calcineurin inhibitor

Clinical trials utilizing regulatory T cell (Treg) therapy in organ transplantation have shown promising results, however, the choice of a standard immunosuppressive regimen is still controversial. Calcineurin inhibitors (CNIs) are one of the most common immunosuppressants for organ transplantation, although they may negatively affect Tregs by inhibiting IL-2 production by conventional T cells. As a strategy to replace IL-2 signaling selectively in Tregs, we have introduced an engineered orthogonal IL-2 (ortho IL-2) cytokine/cytokine receptor (R) pair that specifically binds with each other but does not bind with their wild-type counterparts. Murine Tregs were isolated from recipients and retrovirally transduced with ortho IL-2Rβ during ex vivo expansion. Transduced Tregs (ortho Tregs) were transferred into recipient mice in a mixed hematopoietic chimerism model with tacrolimus administration. Ortho IL-2 treatment significantly increased the ortho IL-2Rβ(+) Treg population in the presence of tacrolimus without stimulating other T cell subsets. All the mice treated with tacrolimus plus ortho IL-2 achieved heart allograft tolerance, even after tacrolimus cessation, whereas those receiving tacrolimus treatment alone did not. These data demonstrate that Treg therapy can be adopted into a CNI-based regimen by utilizing cytokine receptor engineering.

Establishment of operational tolerance to sustain antitumor immunotherapy.

Commonly used immunosuppressive drugs are efficacious in the prevention of transplanted solid organ rejection but, are complicated by an increased rate of malignancies. Treatment of the latter with cancer immunotherapy is frequently associated with increased graft loss from rejection. B16 melanoma was inoculated subcutaneously (s.c.) in the ear of host B6 mice carrying a heterotopic allogenic murine heart. Intratumoral (i.t.) immunotherapy with anti-programmed death-1 (PD-1) and a toll-like receptor 9 (TLR9) agonist was conducted to treat cancer. Cyclosporine A (CsA) therapy or replaced by other immunosuppressive agents was conducted to preserve heart allograft tolerance, respectively. Here we show that long-term allograft-bearing mice receiving CsA therapy and challenged with host-type melanoma resist cancer immunotherapy and reject their grafts after CsA withdrawal. However, when CsA therapy is replaced by intermittent administration of the PI4KIIIβ inhibitor UCB9608, effective antitumor immunity is induced while preserving graft tolerance. UCB9608 switch combined with i.t. immunotherapy resulted in donor-specific tolerance with preserved third-party responses. This operational tolerance may not be dependent on regulatory T cells (Tregs). Immune effects induced by UCB9608 switch following CsA therapy allow for antitumor immunity, opening up new approaches to establish donor-specific operational tolerance in solid organ transplantation with cancer.

Using Selective Bcl2 Inhibition to Induce Cardiac Allograft Tolerance

<h3>Purpose</h3> Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group using a mixed chimerism model with allogeneic bone marrow transplant after non-myeloablative conditioning but not without kidney co-transplantation. B cell lymphoma 2 (Bcl2) inhibition has allowed for durable mixed chimerism with reduced conditioning in the murine skin transplant model. We investigated whether the Bcl2 inhibitor ABT199 could enhance mixed chimerism and induce cardiac allograft tolerance in the absence of kidney cotransplantation. <h3>Methods</h3> Fifteen cynomolgus recipients underwent mixed chimerism conditioning (ATGAM, total body and thymic irradiation) then organ and bone marrow transplantation. Group 1 recipients received heart alone (n=5), Group 2 recipients received heart and kidney cotransplants (n=7), and in Group 3, heart recipients received ABT199 in lieu of kidney cotransplants (n=3).Group 1 and 2 received 3Gy total body irradiation (TBI) while Group 3 received 1.5Gy TBI. All recipients underwent a 28-day course of immunosuppression with a calcineurin inhibitor and anti-CD154 mAb after which all immunosuppression was stopped. <h3>Results</h3> All five Group 1 recipients demonstrated cellular rejection (ISHLT 3R) by 96 days post bone marrow transplant (pBMTx). In contrast, no Group 2 recipients showed early rejection and 6/7 survived >400 days pBMTx. The first two Group 3 recipients developed GvHD and succumbed by days 59 and 68 pBMTx without evidence of rejection. However, after reducing the size of the donor BMT, the third recipient is now 76 days pBMTx with excellent allograft contractility and no evidence of GVHD. Mean peak lymphoid chimerism was 10% on day 26 pBMTx for Group 1, 26% on day 33 pBMTx for Group 2, and 57% on day 54 pBMTx for Group 3. (Figure 1) <h3>Conclusion</h3> After further refinement of the conditioning regimen, ABT199 may allow for the induction of heart allograft tolerance without the need for kidney co-transplantation and with a reduction in TBI thereby improving safety.

Cardiac Allograft Tolerance Induction with Ox40L Costimulatory Blockade

<h3>Purpose</h3> Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group via hematopoietic mixed chimerism with allogeneic bone marrow and kidney co-transplantation. This model has failed without the regulatory mechanisms associated with the donor kidney. We hypothesized that rapamycin and anti-OX40L mAb (KY1005, Kymab Ltd) could substitute for kidney co-transplantation and induce cardiac allograft tolerance by providing a favorable immunologic milieu for regulatory T cells (Tregs). <h3>Methods</h3> Fourteen cynomolgus NHPs underwent mixed chimerism conditioning (ATGAM, total body and thymic irradiation) then organ and bone marrow transplant. Heart Alone recipients received a heart transplant (n=5), Heart/Kidney recipients received a heart and kidney co-transplant (n=7). Heart/OX40L recipients received a heart transplant plus 40 days of anti-OX40L mAb (n=2). All groups underwent a 12 day course of aCD154 mAb. The Heart Alone and Heart Kidney groups received 28 days of cyclosporine while the Heart aOX40L group received 100 days of rapamycin. <h3>Results</h3> All Heart Alone recipients had cellular rejection (ISHLT 3R) by 200 days post bone marrow transplant (pBMT). No Heart/Kidney recipients showed early rejection. Similarly, no Heart/OX40L recipients showed early rejection and one is currently 258 days pBMT without clinical or pathologic evidence of rejection (Figure 1). The other recipient died of viral complications. Mean peak lymphoid chimerism was 10% for the Heart Alone group, 26% for the Heart/Kidney group, and 29% for the Heart/OX40L group. The anti-OX40L protocol also showed an expansion of the Treg population with a peak of 35% Tregs in the CD4+ population. <h3>Conclusion</h3> The OX40L protocol achieved robust lymphoid chimerism with concomitant expansion of Tregs. The surviving Heart/OX40L recipient achieved longer freedom from allograft rejection when compared to the Heart Alone group. OX40L costimulatory blockade may allow for heart allograft tolerance without kidney co-transplantation.

Kidney-induced systemic tolerance of heart allografts in mice

In swine and nonhuman primates, kidney allografts can induce tolerance of heart allografts, leading to their long-term, immunosuppression-free survival. We refer to this phenomenon as kidney-induced cardiac allograft tolerance (KICAT). In this study, we have developed a murine model for KICAT to determine the underlining cellular/molecular mechanisms. Here, we show that spontaneously accepted DBA/2J kidneys in C57BL/6 recipients induce systemic tolerance that results in the long-term acceptance of DBA/2J heart allografts but not third-party cardiac allografts. The state of systemic tolerance of hearts was established 2 weeks after transplantation of the kidney, after which time, the kidney allograft is no longer required. Depletion of Foxp3+ T cells from these mice precipitated rejection of the heart allografts, indicating that KICAT is dependent on Treg function. Acceptance of kidney allografts and cotransplanted heart allografts did not require the thymus. In conclusion, these data show that kidney allografts induce systemic, donor-specific tolerance of cardiac allografts via Foxp3 cells, and that tolerance is independent of the thymus and continued presence of the kidney allograft. This experimental system should promote increased understanding of the tolerogenic mechanisms of the kidney.

CD8-Expressing Transitional B Cells Dominate B Cell Reconstitution after Mixed Chimerism Conditioning for Thoracic Organ Transplantation in Monkeys

Tolerance of heart or lung allografts have been achieved for the first time in nonhuman primates (NHPs) using a mixed chimerism conditioning regimen. However, the mechanisms leading to this state of unresponsiveness remain unclear. Transitional B cells have been recently highlighted for their IL-10-dependent suppressive capabilities and their potential to assist in Treg development and sustainability. Here, we assessed the prevalence of transitional B cells during leukocyte reconstitution in conditioned NHP recipients of combined bone marrow and heart or lung allografts.

Donor-Specific Regulatory T Cells Acquired from Tolerant Mice Bearing Cardiac Allograft Promote Mixed Chimerism and Prolong Intestinal Allograft Survival.

The induction of donor-specific transplant tolerance has always been a central problem for small bowel transplantation (SBT), which is thought to be the best therapy for end-stage bowel failure. With the development of new tolerance-inducing strategies, mixed chimerism induced by co-stimulation blockade has become most potent for tolerance of allografts, such as skin, kidney, and heart. However, a lack of clinically available co-stimulation blockers has hindered efficient application in humans. Furthermore, unlike those for other types of solid organ transplantation, strategies to induce robust mixed chimerism for intestinal allografts have not been fully developed. To improve current mixed chimerism induction protocols for future clinical application, we developed a new protocol using donor-specific regulatory T (Treg) cells from mice with heart allograft tolerance, immunosuppressive drugs which could be used clinically and low doses of irradiation. Our results demonstrated that donor-specific Treg cells acquired from tolerant mice after in vitro expansion generate stable chimerism and lead to acceptance of intestinal allograft. Increased intragraft Treg cells and clonal deletion contribute to the development of SBT tolerance.

Effects of Lung Cotransplantation on Cardiac Allograft Tolerance Across a Full Major Histocompatibility Complex Barrier in Miniature Swine.

A 12-day course of high-dose tacrolimus induces tolerance of major histocompatibility complex-mismatched lung allografts in miniature swine but does not induce tolerance of heart allografts unless a kidney is cotransplanted. To determine whether lungs share with kidneys the ability to induce cardiac allograft tolerance, we investigated heart-lung cotransplantation using the same induction protocol. Hearts (n = 3), heart-kidneys (n = 3), lungs (n = 6), and hearts-lungs (n = 3) were transplanted into fully major histocompatibility complex-mismatched recipients treated with high-dose tacrolimus for 12 days. Serial biopsy samples were used to evaluate rejection, and in vitro assays were used to detect donor responsiveness. All heart-kidney recipients and five of six lung recipients demonstrated long-term graft survival for longer than 272 days, while all heart recipients rejected their allografts within 35 days. Tolerant recipients remained free of alloantibody and showed persistent donor-specific unresponsiveness by cell-mediated lympholysis/mixed-lymphocyte reaction. In contrast, heart-lung recipients demonstrated rejection of both allografts (days 47, 55, and 202) and antidonor responsiveness in vitro. In contrast to kidneys, lung cotransplantation leads to rejection of both heart and lung allografts, indicating that lungs do not have the same tolerogenic capacity as kidneys. We conclude that cells or cell products present in kidney, but not heart or lung allografts, have a unique capacity to confer unresponsiveness on cotransplanted organs, most likely by amplifying host regulatory mechanisms.

Immunomodulatory Strategies Directed Toward Tolerance of Vascularized Composite Allografts.

Achieving tolerance of vascularized composite allografts (VCAs) would improve the risk-to-benefit ratio in patients who undergo this life-enhancing, though not lifesaving, transplant. Kidney cotransplantation along with a short course of high-dose immunosuppression enables tolerance of heart allografts across a full major histocompatibility complex (MHC) mismatch. In this study, we investigated whether tolerance of VCAs across full MHC disparities could be achieved in animals already tolerant of heart and kidney allografts. Miniature swine that were tolerant of heart and/or kidney allografts long term underwent transplantation of myocutaneous VCA across the same MHC barrier. Before VCA transplant, group 1 (n = 3) underwent class I-mismatched kidney transplantation; group 2 (n = 3) underwent 2 sequential class I-mismatched kidney transplantations; group 3 (n = 2) underwent haploidentical MHC-mismatched heart/kidney transplantation; and group 4 (n = 2) underwent full MHC-mismatched heart/kidney transplantation. All 3 animals in group 1 and 2 of 3 animals in group 2 showed skin rejection within 85 days; 1 animal in group 2 showed prolonged skin survival longer than 200 days. Animals in groups 3 and 4 showed skin rejection within 30 days and regained in vitro evidence of donor responsiveness. This is the first preclinical study in which hearts, kidneys, and VCAs have been transplanted into the same recipient. Despite VCA rejection, tolerance of heart and kidney allografts was maintained. These results suggest that regulatory tolerance of skin is possible but not generally achieved by the same level of immunomodulation that is capable of inducing tolerance of heart and kidney allografts. Achieving tolerance of skin may require additional immunomodulatory therapies.

Transient mixed chimerism for allograft tolerance.

Mixed chimerism discovered in Freemartin cattle by Ray Owen 70years ago paved the way for research on immune tolerance. Since his discovery, significant progress has been made in the effort to induce allograft tolerance via mixed chimerism in various murine models. However, induction of persistent mixed chimerism has proved to be extremely difficult in major histocompatibility complex mismatched humans. Chimerism induced in humans tends to either disappear or convert to full donor chimerism, depending on the intensity of the conditioning regimen. Nevertheless, our studies in both NHPs and humans have clearly demonstrated that renal allograft tolerance can be induced by transient mixed chimerism. Our studies have shown that solid organ allograft tolerance via transient mixed chimerism 1) requires induction of multilineage hematologic chimerism, 2) depends on peripheral regulatory mechanisms, rather than thymic deletion, for long-term maintenance, 3) is organ specific (kidney and lung but not heart allograft tolerance are feasible). A major advantage of tolerance induction via transient mixed chimerism is exclusion of the risk of graft-versus-host disease. Our ongoing studies are directed toward improving the consistency of tolerance induction, reducing the morbidity of the conditioning regimen, substituting clinically available agents, such as Belatacept for the now unavailable anti-CD2 monoclonal antibody, and extending the protocol to recipients of deceased donor allografts.

Depletion of Foxp3+ T Cells Abrogates Tolerance of Skin and Heart Allografts in Murine Mixed Chimeras Without the Loss of Mixed Chimerism

The relative contribution of central and peripheral mechanisms to the generation and maintenance of allograft tolerance is of considerable interest. Here, we present new evidence that regulatory T cells (Foxp3(+) ) maintain skin and heart allograft tolerance in mixed hematopoietic chimeric mice. Transient depletion of both donor- and recipient-derived Foxp3(+) cells was necessary and sufficient to induce decisive rejection of long-accepted skin and heart allografts. In contrast, stable hematopoietic chimerism remained, and there was no detectable induction of donor-specific reactivity to hematopoietic cells. Foxp3(+) cell depletion did not result in the rejection of skin grafts of only MHC-disparate donors (B6.C-H2(d) /bByJ), indicating that MHC antigens were not the target in the graft. We conclude that two different mechanisms of tolerance are present in mixed chimeras. Hematopoietic chimerism, resistant to Foxp3(+) depletion, is probably due to deletional tolerance to MHC antigens, as supported by previous studies. In contrast, regulatory tolerance mechanisms involving Foxp3(+) cells are required to control reactivity against non-MHC antigens not present on hematopoietic lineages.

B7-H1 Expression Is Critical to Cardiac Allograft Tolerance Induced By the Combination Therapy of Mesenchymal Stem Cells and Rapamycin.

Background: Mesenchymal stem cells (MSCs) become an attractive tool to suppress immune response. We have previously reported that MSC-based therapy in combination with rapamycin induced cardiac allograft tolerance in a mouse model. The present study investigated the role of MSC-expressing B7-H1 in MSC-mediated cardiac allograft protection. Methods & Results:MSCs (1×106, i.v.) from C57BL/6 donor mice were injected into BALB/c recipients 24 hours after receiving a heterotopic C57BL/6 heart graft. Grafts in untreated recipients were rapidly rejected in 7.5±0.7 days. Either MSCs or rapamycin monotherapy doubled allograft survival time to 15.8±1.5 days and 16.3±1.1 days, respectively. MSCs in combination with rapamycin induced heart allograft tolerance with normal graft histology and achieved graft mean survival time (MST) for more than 100 days. In contrast, treatment with B7-H1-blocking MSCs was not able to prolong allograft survival (MST: 8.7±1.1 days), and in combination with rapamycin prevented tolerance induction. Graft MST in the recipients treated with B7-H1-blocking MSC-based combination therapy was significantly reduced to 18.5±0.8 days. In addition, Flow cytomytry analysis showed that B7-H1 is required for MSC-mediated downregulation of antibody production and enhancement of the frequency of tolerogeneic dendritic cells (Tol-DCs) and CD4+CD25+Foxp3+ cells in tolerant recipients. In vivo treatment with MSCs expressing B7-H1 is also involved in the modulation of CD83 and cytokine (IL-4 and IL-10) expression of B cells. Furthermore, MSC-mediated suppression of B cells measured by MLR, ELISA and transwell assays requires B7-H1 and contact between CD19+ B cells and MSCs. Conclusion:This study supports a critical role of B7-H1 in MSC-mediated immunosuppression and tolerance induction in transplantation, and would clearly emphasize the significant clinical potential of MSCs.

Heart Allograft Tolerance Induced and Maintained by Vascularized Hind-Limb Transplant in Rats

Organ/tissue transplantation has become an effective therapy for end-stage diseases. However, immunosuppression after transplantation may cause severe side effects. Donor-specific transplant tolerance was proposed to solve this problem. In this study, we report a novel method for inducing and maintaining heart allograft tolerance rats. First, we induced indefinite vascularized hind-limb allograft survival with a short-term antilymphocyte serum + Cyclosporine A treatment. Peripheral blood chimerism disappeared 6-7 weeks after immunosuppression was withdrawn. Then the recipients accepted secondary donor-strain skin and heart transplantation 200 days following vascularized hind-limb transplantation without any immunosuppression, but rejected third party skin allografts, a status of donor-specific tolerance. The ELISPOT results suggested a mechanism of clone deletion. These findings open new perspectives for the role of vascularized hind-limb transplant in the induction and maintenance of organ transplantation tolerance.

Immunology of Vascularized Composite Allografts

Immunology of Vascularized Composite Allografts

Effect of Kidney Cotransplantation on Induction of Heart Graft Tolerance in Nonhuman Primates (NHPs)

Introduction: Tolerance has been achieved in NHPs and human recipients of kidney allogeneic transplants through induction of transient hematopoietic mixed chimerism. However, the same approach has failed to induce tolerance of cardiac allografts in primates. In this study, we investigated whether immune regulation induced by kidney allografts in this model could spread to heart transplants from the same donor thereby preventing their rejection in cynomolgus monkeys. Methods: Cynomolgus monkeys underwent donor bone marrow transplantation (DBMT) and either heart/kidney co-transplantation (n=6) or heart transplant alone (n=4) following nonmyeloablative conditioning, which consisted of TBI (1.5 Gy ×2), thymic irradiation (7Gy), horse ATG, anti-CD154 mAb, and a 28-day course of cyclosporine. One monkey received DBMT 4 months after co-transplantation. Results: The majority of recipients of DBMT and co-transplantation of the heart and kidney achieved long-term survival (380*, 391*, >366, >261, 131, >51, >30 days; *[heart rejection after removal of the kidney on day 315]) of both allografts without maintenance immunosuppression. Critical involvement of the kidney allograft in the maintenance of tolerance was clearly demonstrated as heart allograft was rejected shortly after removal of the kidney allograft. In stark contrast, all four monkeys transplanted with an isolated heart acutely rejected shortly after cessation of Cyclosporine. IFNγ-ELISPOT and MLR assays in tolerant recipients showed donor specific hyporesponsiveness. T regulatory cells were found enriched in 7-8 times more in the kidney allograft comparing with those in the peripheral blood. Conclusion: This is the first demonstration of heart allograft tolerance in nonhuman primates. Apparently, the presence of the allogeneic kidney is necessary for the maintenance of tolerance of the transplanted heart. The mechanistic studies suggested that the kidney allograft facilitates to induce/expand specific regulatory T cells induced after transient mixed chimerism.Figure: [Cardiac Graft Survival]

Regulatory T Cells Are Critical to Tolerance Induction in Presensitized Mouse Transplant Recipients Through Targeting Memory T Cells

Memory T cells are a significant barrier to induction of transplant tolerance. However, reliable means to target alloreactive memory T cells have remained elusive. In this study, presensitization of BALB/c mice with C57BL/6 skin grafts generated a large number of OX40(+)CD44(hi)effector/memory T cells and resulted in rapid rejection of donor heart allografts. Recognizing that anti-OX40L monoclonal antibody (mAb) (alpha-OX40L) monotherapy prolonged graft survival through inhibition and apoptosis of memory T cells in presensitized recipients, alpha-OX40L was added to the combined treatment protocol of LF15-0195 (LF) and anti-CD45RB (alpha-CD45RB) mAb-a protocol that induced heart allograft tolerance in non-presensitized recipients but failed to induce tolerance in presensitized recipients. Interestingly, this triple therapy restored donor-specific heart allograft tolerance in our presensitized model that was associated with induction of tolerogenic dendritic cells and CD4(+)CD25(+)Foxp3(+) T regulatory cells (Tregs). Of note, CD25(+) T cell depletion in triple therapy recipients prevented establishment of allograft tolerance. In addition, adoptive transfer of donor-primed effector/memory T cells into tolerant recipients markedly reduced levels of Tregs and broke tolerance. Our findings indicated that targeting memory T cells, by blocking OX40 costimulation in presensitized recipients was very important to expansion of Tregs, which proved critical to development of tolerance.