MHC-mismatched Cardiac Allografts Research Articles

Deleting Runx3 in CD4+ T Cells Disrupts Effector Functions and Facilitates Tolerance Induction in Solid Organ Transplantation

Abstract Long-term transplantation tolerance depends on the control of graft-reactive T cells. In particular, the alloreactivity of CD4+ T cells needs to be suppressed as these cells are both necessary and sufficient for cardiac allograft rejection. Although experimental protocols such as anti-CD154-mediated co-stimulation blockade can stably induce donor-specific tolerance in MHC-mismatched cardiac allografts, other more immunogenic organs such as skin are less susceptible to tolerogenic treatments, in part due to additional reactivity of the host against donor commensals that accompany the graft. The transcriptional mechanisms that enable CD4+ T cells to drive rejection and whose targeting can promote tolerance are not well understood. RNAseq comparison of the transcriptome in graft-reactive T cells from rejecting versus tolerant heart allograft recipients revealed several transcription factors differentially expressed between rejected and tolerant mice in both spleen and graft. Here, we report that the transcription factor Runx3 in alloreactive CD4+ T cells promotes Th1 and cytotoxic response against the allograft and resistance to tolerance induction. Conversely, ablating Runx3 in alloreactive CD4+ T cells augments the efficacy of anti-CD154 blockade treatment enabling skin graft acceptance.

T Cell-Specific Adaptor Protein Regulates Mitochondrial Function and CD4+ T Regulatory Cell Activity In Vivo following Transplantation.

The T cell-specific adaptor protein (TSAd), encoded by the SH2D2A gene, is an intracellular molecule that binds Lck to elicit signals that result in cytokine production in CD4+ T effector cells (Teff). Nevertheless, using Sh2d2a knockout (KO; also called TSAd-/-) mice, we find that alloimmune CD4+ Teff responses are fully competent in vivo. Furthermore, and contrary to expectations, we find that allograft rejection is accelerated in KO recipients of MHC class II-mismatched B6.C-H-2bm12 heart transplants versus wild-type (WT) recipients. Also, KO recipients of fully MHC-mismatched cardiac allografts are resistant to the graft-prolonging effects of costimulatory blockade. Using adoptive transfer models, we find that KO T regulatory cells (Tregs) are less efficient in suppressing Teff function and they produce IFN-γ following mitogenic activation. In addition, pyrosequencing demonstrated higher levels of methylation of CpG regions within the Treg-specific demethylated region of KO versus WT Tregs, suggesting that TSAd, in part, promotes Treg stability. By Western blot, Lck is absent in the mitochondria of KO Tregs, and reactive oxygen species production by mitochondria is reduced in KO versus WT Tregs. Full transcriptomic analysis demonstrated that the key mechanism of TSAd function in Tregs relates to its effects on cellular activation rather than intrinsic effects on mitochondria/metabolism. Nevertheless, KO Tregs compensate for a lack of activation by increasing the number of mitochondria per cell. Thus, TSAd serves as a critical cell-intrinsic molecule in CD4+Foxp3+ Tregs to regulate the translocation of Lck to mitochondria, cellular activation responses, and the development of immunoregulation following solid organ transplantation.

Peritransplant VLA-4 blockade inhibits endogenous memory CD8 T cell infiltration into high-risk cardiac allografts and CTLA-4Ig resistant rejection.

Recipient endogenous memory CD8 T cells expressing reactivity to donor class I MHC infiltrate MHC-mismatched cardiac allografts within 24hours after reperfusion and express effector functions mediating graft injury. The current study tested the efficacy of Very Late Antigen-4 (VLA-4) blockade to inhibit endogenous memory CD8 T cell infiltration into cardiac allografts and attenuate early posttransplant inflammation. Peritransplant anti-VLA-4 mAb given to C57BL6 (H-2b ) recipients of AJ (H-2a ) heart allografts completely inhibited endogenous memory CD4 and CD8 T cell infiltration with significant decrease in macrophage, but not neutrophil, infiltration into allografts subjected to either minimal or prolonged cold ischemic storage (CIS) prior to transplant, reduced intra-allograft IFN-γ-induced gene expression and prolonged survival of allografts subjected to prolonged CIS in CTLA-4Ig treated recipients. Anti-VLA-4 mAb also inhibited priming of donor-specific T cells producing IFN-γ until at least day 7 posttransplant. Peritransplant anti-VLA plus anti-CD154 mAb treatment similarly prolonged survival of allografts subjected to minimal or increased CIS prior to transplant. Overall, these data indicate that peritransplant anti-VLA-4 mAb inhibits early infiltration memory CD8 T cell infiltration into allografts with a marked reduction in early graft inflammation suggesting an effective strategy to attenuate negative effects of heterologous alloimmunity in recipients of higher risk grafts.

Graft protective effect and induction of CD4+Foxp3+ cell by Thrombomodulin on allograft arteriosclerosis in mice

BackgroundThrombomodulin (TM) is a promising therapeutic natural anti-coagulant, which exerts the effects to control disseminated intravascular coagulation. However, little is known whether TM on micro-vessels could play an important role in the regulation of intimal hyperplasia. We investigated the vessel-protective effect of TM in the survival of fully major histocompatibility complex (MHC)-mismatched murine cardiac allograft transplantation.MethodsCBA recipients transplanted with a C57BL/6 heart received intraperitoneal administration of normal saline or 0.2, 2.0, and 20.0 μg/day of TM for 7 days (n = 5, 7, 11, and 11, respectively). Immunohistochemical and fluorescent staining studies were performed to determine whether CD4+Foxp3+ regulatory T cell were generated at 2 and 4 weeks after grafting. Morphometric analysis for neointimal formation in the coronary arteries of the transplanted allograft was conducted at 2 and 4 weeks after grafting.ResultsUntreated CBA recipients rejected C57BL/6 cardiac grafts acutely (median survival time [MST], 7 days). CBA recipients exposed with the above doses had significantly prolonged allograft survival (MSTs, 17, 24 and 50 days, respectively). Morphometric assessment showed that intimal hyperplasia was clearly suppressed in the left and right coronary arteries or allografts from TM-exposed recipients 2 and 4 weeks. Immunohistochemical studies at 2 weeks showed more CD4+Foxp3+ cells and lower myocardial damage in the allografts from TM-exposed recipients. Notably, fluorescent staining studies demonstrated that TM-exposed recipients 4 weeks post-engraftment had strong aggregation of CD4+Foxp3+ cells in the intima of the coronary arteries of the cardiac allografts.ConclusionsTM may prolong the survival of fully MHC-mismatched cardiac allografts through suppressing intimal hyperplasia and inducing the accumulation of regulatory CD4+Foxp3+ cells within coronary arteries.

Allograft dendritic cell p40 homodimers activate donor-reactive memory CD8+ T cells.

Recipient endogenous memory T cells with donor reactivity pose an important barrier to successful transplantation and costimulatory blockade-induced graft tolerance. Longer ischemic storage times prior to organ transplantation increase early posttransplant inflammation and negatively impact early graft function and long-term graft outcome. Little is known about the mechanisms enhancing endogenous memory T cell activation to mediate tissue injury within the increased inflammatory environment of allografts subjected to prolonged cold ischemic storage (CIS). Endogenous memory CD4+ and CD8+ T cell activation is markedly increased within complete MHC-mismatched cardiac allografts subjected to prolonged versus minimal CIS, and the memory CD8+ T cells directly mediate CTLA-4Ig-resistant allograft rejection. Memory CD8+ T cell activation within allografts subjected to prolonged CIS requires memory CD4+ T cell stimulation of graft DCs to produce p40 homodimers, but not IL-12 p40/p35 heterodimers. Targeting p40 abrogates memory CD8+ T cell proliferation within the allografts and their ability to mediate CTLA-4Ig-resistant allograft rejection. These findings indicate a critical role for memory CD4+ T cell-graft DC interactions to increase the intensity of endogenous memory CD8+ T cell activation needed to mediate rejection of higher-risk allografts subjected to increased CIS.

Aquaporin 4 blockade improves survival of murine heart allografts subjected to prolonged cold ischemia.

Prolonged cold ischemia storage (CIS) is a leading risk factor for poor transplant outcome. Existing strategies strive to minimize ischemia-reperfusion injury in transplanted organs, yet there is a need for novel approaches to improve outcomes of marginal allografts and expand the pool of donor organs suitable for transplantation. Aquaporins (AQPs) are a family of water channels that facilitate homeostasis, tissue injury, and inflammation. We tested whether inhibition of AQP4 improves the survival of fully MHC-mismatched murine cardiac allografts subjected to 8hours of CIS. Administration of a small molecule AQP4 inhibitor during donor heart collection and storage and for a short-time posttransplantation improves the viability of donor graft cells, diminishes donor-reactive T cell responses, and extends allograft survival in the absence of other immunosuppression. Furthermore, AQP4 inhibition is synergistic with cytotoxic T lymphocyte-associated antigen 4-Ig in prolonging survival of 8-hour CIS heart allografts. AQP4 blockade markedly reduced T cell proliferation and cytokine production in vitro, suggesting that the improved graft survival is at least in part mediated through direct effects on donor-reactive T cells. These results identify AQPs as a promising target for diminishing donor-specific alloreactivity and improving the survival of high-risk organ transplants.

Histone/protein deacetylase 11 targeting promotes Foxp3+ Treg function

Current interest in Foxp3+ T-regulatory (Treg) cells as therapeutic targets in transplantation is largely focused on their harvesting pre-transplant, expansion and infusion post-transplantation. An alternate strategy of pharmacologic modulation of Treg function using histone/protein deacetylase inhibitors (HDACi) may allow more titratable and longer-term dosing. However, the effects of broadly acting HDACi vary, such that HDAC isoform-selective targeting is likely required. We report data from mice with constitutive or conditional deletion of HDAC11 within Foxp3+ Treg cells, and their use, along with small molecule HDAC11 inhibitors, in allograft models. Global HDAC11 deletion had no effect on health or development, and compared to WT controls, Foxp3+ Tregs lacking HDAC11 showed increased suppressive function, and increased expression of Foxp3 and TGF-β. Likewise, compared to WT recipients, conditional deletion of HDAC11 within Tregs led to long-term survival of fully MHC-mismatched cardiac allografts, and prevented development of transplant arteriosclerosis in an MHC class II-mismatched allograft model. The translational significance of HDAC11 targeting was shown by the ability of an HDAC11i to promote long-term allograft allografts in fully MHC-disparate strains. These data are powerful stimuli for the further development and testing of HDAC11-selective pharmacologic inhibitors, and may ultimately provide new therapies for transplantation and autoimmune diseases.

Simvastatin Treatment Upregulates Anti-Fibrotic Bone Morphogenetic Protein-7 Expression at Rat Cardiac Allograft Rejection

Background: Bone morphogenetic protein (BMP)-7 mediates ischemic tolerance and anti-fibrotic effects in various organs such as kidney and heart. Recently, reno- and podocyte-protective effects of a potent HMG-CoA reductase inhibitor, pitavastatin, were accompanied by BMP-7 upregulation. Methods: Here, we investigated the effect of simvastatin treatment on BMP-7 expression in major MHC-mismatched rat cardiac allografts subjected to ischemia-reperfusion injury and adaptive immune activation at 10 days. Results: We localized Smad2 activity and Reca-1⁺ fibroblast specific protein-1⁺ immunoreactivity, suggesting endothelial-to-mesenchymal transition, at fibrotic borderline of cardiac allografts at 10 days. Simvastatin donor and recipient combination treatment significantly upregulated cardiac allograft BMP-7 expression when compared to nontreated controls at 10 days. Conclusion: The beneficial effect of statin treatment on cardiac allograft may in part be mediated through the upregulation of BMP-7.

Tracking of TCR-Transgenic T Cells Reveals That Multiple Mechanisms Maintain Cardiac Transplant Tolerance in Mice.

Solid organ transplantation tolerance can be achieved following select transient immunosuppressive regimens that result in long-lasting restraint of alloimmunity without affecting responses to other antigens. Transplantation tolerance has been observed in animal models following costimulation or coreceptor blockade therapies, and in a subset of patients through induction protocols that include donor bone marrow transplantation, or following withdrawal of immunosuppression. Previous data from our lab and others have shown that proinflammatory interventions that successfully prevent the induction of transplantation tolerance in mice often fail to break tolerance once it has been stably established. This suggests that established tolerance acquires resilience to proinflammatory insults, and prompted us to investigate the mechanisms that maintain a stable state of robust tolerance. Our results demonstrate that only a triple intervention of depleting CD25+ regulatory T cells (Tregs), blocking programmed death ligand-1 (PD-L1) signals, and transferring low numbers of alloreactive T cells was sufficient to break established tolerance. We infer from these observations that Tregs and PD-1/PD-L1 signals cooperate to preserve a low alloreactive T cell frequency to maintain tolerance. Thus, therapeutic protocols designed to induce multiple parallel mechanisms of peripheral tolerance may be necessary to achieve robust transplantation tolerance capable of maintaining one allograft for life in the clinic.

Inhibition of Endogenous Memory CD8 T Cell Infiltration into Cardiac Allografts Subjected to Prolonged Cold Ischemic Storage is Required to Promote Long-Term Graft Survival Naoki Kohei, Hidetoshi

Abstract Endogenous memory CD8 T cells infiltrate MHC-mismatched cardiac allografts and produce IFN-γ in response to donor class I MHC within 24 hrs after graft reperfusion in mice. We have reported that prolonged cold ischemic graft storage (CIS) provokes intense inflammation within hours after allograft reperfusion and promotes CTLA-4Ig-resistant endogenous memory CD8 T cell rejection of the allograft by day 28 post-transplant (whereas CTLA-4Ig prolongs survival of allografts subjected to minimal CIS > day 60). We tested strategies inhibiting endogenous memory CD8 T cell infiltration into cardiac allografts subjected to prolonged CIS to promote long-term allograft survival. Peri-transplant anti-LFA-1 mAb and anti-CD154 mAb treatment of recipients of allografts subjected to minimal CIS prolonged survival of 60% of allograft > 100 days. In contrast, this treatment prolonged only 20% of allografts subjected to prolonged CIS beyond day 80 post-transplant, with rejection accompanied by the appearance of high titers of donor-specific antibody (DSA >10,000 vs. <100 in long-term survivors). Use of a new regimen: peri-transplant recipient treatment with anti-LFA-1 mAb, anti-TNFa mAb, and anti-CD154 mAb plus additional doses of anti-CD154 mAb on days 14 and 16 post-transplant promoted long-term survival of 60% allografts subjected to prolonged CIS past day 100 post-transplant. Recipients with surviving allografts accepted donor skin allografts, but rejected third-party skin allografts. These studies indicate long-term survival of allografts subjected to prolonged CIS by using strategies that include reagents inhibiting endogenous memory CD8 T cell infiltration into the allografts and reagents that inhibit the production of DSA.

IL-1 Receptor Signaling on Graft Parenchymal Cells Regulates Memory and De Novo Donor-Reactive CD8 T Cell Responses to Cardiac Allografts

Reperfusion of organ allografts induces a potent inflammatory response that directs rapid memory T cell, neutrophil, and macrophage graft infiltration and their activation to express functions mediating graft tissue injury. The role of cardiac allograft IL-1 receptor (IL-1R) signaling in this early inflammation and the downstream primary alloimmune response was investigated. When compared with complete MHC-mismatched wild-type cardiac allografts, IL-1R(-/-) allografts had marked decreases in endogenous memory CD8 T cell and neutrophil infiltration and expression of proinflammatory mediators at early times after transplant, whereas endogenous memory CD4 T cell and macrophage infiltration was not decreased. IL-1R(-/-) allograft recipients also had marked decreases in de novo donor-reactive CD8, but not CD4, T cell development to IFN-γ-producing cells. CD8 T cell-mediated rejection of IL-1R(-/-) cardiac allografts took 3 wk longer than wild-type allografts. Cardiac allografts from reciprocal bone marrow reconstituted IL-1R(-/-)/wild-type chimeric donors indicated that IL-1R signaling on graft nonhematopoietic-derived, but not bone marrow-derived, cells is required for the potent donor-reactive memory and primary CD8 T cell alloimmune responses observed in response to wild-type allografts. These studies implicate IL-1R-mediated signals by allograft parenchymal cells in generating the stimuli-provoking development and elicitation of optimal alloimmune responses to the grafts.

Induction of Regulatory T Cells and Prolongation of Fully Allogeneic Cardiac Grafts by Herbal Medicine, Shohangekabukuryo-to

Objective: Shohangekabukuryo-to (Tsumura Japan [TJ]-21) is well known as an herbal medicine with few side effects to treat vomiting, nausea, acute and chronic gastritis, and hyperemesis of pregnancy in Japan. In this study, we performed fully allogeneic cardiac grafts in mice to investigate the effect of TJ-21 on alloimmune responses. Methods: CBA (H2k) mice underwent transplantation of a C57BL/6 (H2b) heart and received oral administration of 0.02, 0.2 and 2 g/kg per day of TJ-21 from the day of operation until 7 days afterward. Adoptive transfer study was performed to determine whether regulatory cells were generated. Histologic and cell proliferation studies, flow cytometry analyses and cytokine measurements were also performed. Results: Compared to untreated CBA mice rejected C57BL/6 hearts acutely (median survival time [MST], 8 days), MST of allografts from TJ-21-treated mice was prolonged (MST, 77 days). Moreover, adoptive transfer of whole splenocytes from TJ-21-treated allograft CBA recipients had prolongation of allograft survival in secondary recipients (MST, 53 days). Flow cytometry study showed TJ-21-treated mice had an increased CD4+CD25+Foxp3+ cell population. Cell proliferation showed that interleukin-2 (IL-2) and interferon-γ were inhibited in TJ-21-treated mice, whereas IL-4 and IL-10 were increased. Conclusions: In summary, TJ-21 could induce hyporesponsiveness of fully MHC-mismatched cardiac allografts and generate regulatory cells.

Combination of Anti-BTLA Antibody (6B2) and Anti-PD-1 Antibody (PIM-2) Induced Indefinite Survival of Fully MHC-Mismatched Murine Cardiac Allograft.

Background: Programmed death (PD)-1 and B and T lymphocyte attenuator (BTLA) had been implicated in the regulation of autoimmune and may potentially play an important role in alloimmune responses. In this study, we investigated the effect of anti-PD-1 monoclonal antibody (mAb) (PIM-2) and anti-BTLA mAb (6B2) in fully MHC-mismatched murine model of cardiac allograft transplantation. Methods: CBA mice underwent transplantation of C57BL/6 hearts and received a single dose (100μg) of 6B2 on the day of transplantation (day 0) or four doses on day 0, 3, 6 and 9. Moreover, CBA recipients were also given one dose of combination of 6B2 and PIM-2 on day 0. Adoptive transfer was performed to determine whether regulatory cells were generated. Cell-proliferation and cytokine assessments were also performed. Result: Untreated CBA mice rejected C57BL/6 allografts acutely (median survival time [MST], 7 days). When CBA recipients were given one and four doses of 6B2, the allograft survivals were significantly prolonged (MSTs, 46 and >100 days, respectively). Moreover, when CBA mice were given with combination for one dose of 6B2 and PIM-2, the allograft survival was indefinitely prolonged (MST, >100 days). Secondary CBA recipients given whole splenocytes from primary combination-treated CBA recipients with C57BL/6 cardiac allografts 30 days after grafting had prolonged C57BL/6 allograft survival (MST, >30 days). Proliferation of splenocytes and interferon-γ production were suppressed and interleukin-4 production was increased in combination-treated mice. Conclusion: Combination of anti-BTLA mAb (6B2) and anti-PD-1 mAb (PIM-2) could induce hyporesponsiveness of fully MHC-mismatched cardiac allografts and may generate regulatory cells.

Absence of Allograft TLR4 Receptor Signaling Attenuates the Increased Early Inflammation in Allografts Subjected to Prolonged Cold Ischemic Storage.

Endogenous memory CD8 T cells infiltrate MHC-mismatched cardiac allografts and produce IFN-γ in response to donor class I MHC within 24 hrs after graft reperfusion in mice. Our recent studies have indicated that prolonged cold ischemic storage provokes intense inflammation within hours after allograft reperfusion and promotes endogenous memory CD8 T cell rejection of the allograft. This study tested the role of graft TLR4 signaling in the early post-transplant inflammation and downstream alloimmune response to allografts subjected to prolonged cold ischemic storage. Wild type (WT) BALBc (H-2d) or BALB/c.TLR4-/-hearts were subjected to 8 hours of cold ischemic storage in University of Wisconsin solution and then transplanted to C57BL/6 (H-2b) mice. CD4 and CD8 T cell, macrophage, and neutrophil infiltration into allografts on day 2 post-transplant was assessed by flow cytometry and immunohistochemistry. Levels of mRNA encoding inflammatory cytokines were measured by qPCR. Marked decreases in neutrophil, but not CD4 and CD8 T cell and macrophage, infiltration were observed in TLR4-/- vs. WT BALB/c allografts on day 2 post-transplant. This decrease was associated with marked decreases in the intragraft mRNA expression of the neutrophil chemoattractants CXCL1 and CXCL2 on day 2. The absence of allograft TLR4 signaling did not affect intragraft mRNA expression of CXCL10 and the acute phase cytokines, TNF-α and IL-1β. On day 7, TLR4-deficient allografts had virtually no infiltrating CD4 T cells, macrophages or neutrophils and decreased CD8 T cell infiltration that was accompanied by decreases in mRNA expression in perforin, granzyme B and FasL. These data indicate that the absence of TLR4 signaling in allografts subjected to prolonged cold ischemic storage inhibits the early post-reperfusion inflammation and inflammation at times post-transplant when wild-type allografts are undergoing acute rejection. The results suggest that therapeutic targeting of the TLR4 will reduce the negative impact of early inflammatory events on allograft outcome.

VLA-4/VCAM-1 Interactions Are Required for Endogenous Memory CD8 T Cell Mediated Cardiac Allograft Rejection.

We recently reported that endogenous memory CD8 T cells infiltrate MHC-mismatched cardiac allografts and are activated to produce IFN-γ in response to donor class I MHC molecules within 24 hours after reperfusion in mice and reject allografts subjected to prolonged ischemic storage in the absence of de novo priming of donor-reactive effector T cells. The current study tested the efficacy of anti-VLA-4 mAb to inhibit this early CD8 T cell allograft infiltration and activation during cardiac allograft rejection. Syngeneic or A/J (H-2a) hearts subjected to 0.5 or 8 hrs of cold ischemic storage were transplanted to C57BL/6 (H-2b) recipients. Anti-VLA-4 mAb (200 μg) was given on days -1 and 0. CD4 and CD8 T cell, macrophage, and neutrophil infiltration into grafts was assessed on day 2 post-transplant by flow cytometry and immunohistochemistry. Levels of mRNA encoding inflammatory cytokines were measured by qPCR. Donor-reactive T cell priming to IFN-γ and IL-2 producing cells in recipient spleens was assessed by ELISPOT assay. Recipient treatment with anti-VLA-4 mAb decreased CD4 and CD8 T cell and macrophage, but not neutrophil, infiltration into allografts by 70% of that observed into allografts from control IgG-treated recipients on day 2 post-transplant and completely inhibited donor-reactive CD4 and CD8 T cell priming to IFN-γ and IL-2 producing cells until day 14. Anti-VLA-4 mAb significantly reduced intragraft mRNA levels of CXCL9, CXCL10, and IFN-γ, but not the acute phase cytokines, TNF-α, IL-1β and IL-6, on day 2 post-transplant. These effects led to substantial prolongation in allograft survival (MST: day 12.5 vs day 7.5 in control-Ig treated recipients, p < 0.01). In recipients given allografts subjected to prolonged cold ischemic storage, anti-VLA-4 mAb treatment inhibited endogenous memory CD8 T cell infiltration into allografts and the inflammatory response and significantly prolonged allograft survival. These data indicate that peri-transplant anti-VLA-4 mAb inhibits endogenous memory CD8 T-cells infiltration into allografts subjected to prolonged cold ischemic storage and the accompanying inflammation. Therapeutic targeting of this pathway will reduce the negative impact of early memory CD8 T cell-dependent inflammatory events on graft outcome that will be particularly important for recipients with high frequencies of donor-reactive memory CD8 T cells.

Absence of Allograft Interleukin (IL)-1 Receptor Signaling Attenuates Early Graft Inflammation and Donor-Reactive CD8 T Cell Priming.

Endogenous memory CD8 T cells infiltrate MHC-mismatched cardiac allografts and produce IFN-γ in response to donor class I MHC within 24 hrs after graft reperfusion in mice. This study tested the role of graft IL-1 receptor signaling in the early post-transplant inflammation and downstream alloimmune response to the allograft. Wild type (WT) C57BL/6 (H-2b) or B6.IL1R-/-hearts were transplanted to A/J (H-2a) mice. CD4 and CD8 T cell, macrophage, and neutrophil infiltration into allografts on day 2 post-transplant was assessed by flow cytometry and immunohistochemistry. Levels of mRNA encoding inflammatory cytokines were measured by qPCR. Donor-reactive T cell priming to IFN-γ producing cells in recipient spleens was assessed by ELISPOT assay. Marked decreases in CD8 T cell and neutrophil, but not CD4 T cell and macrophage, infiltration were observed in B6.IL-1R-/- vs. WT B6 allografts on day 2 post-transplant. The absence of allograft IL-1R signaling also resulted in significant decreases in intragraft mRNA expression of CXCL9, CXCL10, IFN-γ, FasL, granzyme B, perforin and the acute phase cytokines, TNF-α, IL-1β and IL-6. Whereas donor-reactive CD4 T cell priming to IFN-g producing cells in response to B6.IL-1R-/- and WT B6 allografts was similar, donor-reactive CD8 T cell priming was low-absent in response to B6.IL-1R-/- allografts until day 11. These effects resulted in modest but significant prolongation of B6.IL-1R-/- allograft survival (MST: day 11.5 vs day 7.5 for WT B6 allografts, p < 0.05). A/J mice depleted of CD8 T cells rejected WT B6 and B6.IL-1R-/- allografts by day 15. A/J mice depleted of CD4 T cells rejected WT B6 cardiac allografts by day 25, whereas CD4 depletion promoted long term survival of the B6.IL-1R-/- allografts. These data indicate that the absence of IL-1R signaling in allografts inhibits early endogenous memory CD8 T cell infiltration into the allograft and the accompanying inflammation. The results suggest that therapeutic targeting of this pathway will reduce the negative impact of early memory CD8 T cell-dependent inflammatory events that will be particularly important for recipients with high frequencies of donor-reactive memory CD8 T cells.

Angiopoietin-2 Inhibition Prevents Transplant Ischemia-Reperfusion Injury and Chronic Rejection in Rat Cardiac Allografts

Transplant ischemia-reperfusion injury (Tx-IRI) and allograft dysfunction remain as two of the major clinical challenges after heart transplantation. We investigated the role of angiopoietin-2 (Ang2) in Tx-IRI and rejection using fully MHC-mismatched rat cardiac allografts. We report that plasma levels of Ang2 were significantly enhanced in the human and rat recipients of cardiac allografts, but not in the rat recipients of syngrafts, during IRI. Ex vivo intracoronary treatment of rat cardiac allografts with anti-Ang2 antibody before 4-h cold preservation prevented microvascular dysfunction, endothelial cell (EC) adhesion molecule expression and leukocyte infiltration, myocardial injury and the development of cardiac fibrosis and allograft vasculopathy. Recipient preoperative and postoperative treatment with anti-Ang2 antibody produced otherwise similar effects without effecting microvascular dysfunction, and in additional experiments prolonged allograft survival. Recipient preoperative treatment alone failed to produce these effects. Moreover, ex vivo intracoronary treatment of allografts with recombinant Ang2 enhanced Tx-IRI and, in an add-back experiment, abolished the beneficial effect of the antibody. We demonstrate that neutralization of Ang2 prevents EC activation, leukocyte infiltration, Tx-IRI and the development of chronic rejection in rat cardiac allografts. Our results suggest that blocking Ang2 pathway is a novel, clinically feasible, T cell-independent strategy to protect cardiac allografts.

Endogenous Memory CD8 T Cells Are Activated Within Cardiac Allografts Without Mediating Rejection

Endogenous memory CD8 T cells infiltrate MHC-mismatched cardiac allografts within 12–24 h posttransplant in mice and are activated to proliferate and produce IFN-γ. To more accurately assess the graft injury directly imposed by these endogenous memory CD8 T cells, we took advantage of the ability of anti-LFA-1 mAb given to allograft recipients on days 3 and 4 posttransplant to inhibit the generation of primary effector T cells. When compared to grafts from IgG-treated recipients on day 7 posttransplant, allografts from anti-LFA-1 mAb-treated recipients had increased numbers of CD8 T cells but these grafts had marked decreases in expression levels of mRNA encoding effector mediators associated with graft injury and decreases in donor-reactive CD8 T cells producing IFN-γ. Despite this decreased activity within the allograft, CD8 T cells in allografts from recipients treated with anti-LFA-1 mAb continued to proliferate up to day 7 posttransplant and did not upregulate expression of the exhaustion marker LAG-3 but did have decreased expression of ICOS. These results indicate that endogenous memory CD8 T cells infiltrate and proliferate in cardiac allografts in mice but do not express sufficient levels of functions to mediate overt graft injury and acute rejection.

Alloantigen-Driven Regulatory T Cells Are More Effective at Regulating Rejection in Immunocompetent Recipients than Freshly Isolated or Expanded nTreg

Introduction: Regulatory T cells (Treg) are being considered for evaluation as a cellular therapy in clinical transplantation and most suggested protocols will examine polyclonally-expanded naturally occurring populations (nTreg). Whilst both mouse and human nTreg can regulate rejection in adoptive transfer mouse models, it is less clear whether they have the same impact in immunocompetent recipients, a question of considerable relevance to the clinical situation. Using an approach that enriches for alloantigen-experienced Treg in vitro, we have been able to compare directly the effectiveness of freshly isolated nTreg, expanded nTreg and alloantigen-driven adaptive Treg in immunocompetent mouse heart allograft recipients. Methods: Recipient (CBA H-2k) splenic CD4+CD25+ nTreg were isolated by MACS (purity >95% CD25+) and were used either after isolation or expanded with plate-bound anti-CD3 and anti-CD28 antibody plus IL-2 for 5 days. Antigen-experienced Treg (IFN-γ Treg) were enriched in vitro by stimulating naïve CBA CD4+ T cells with modulated donor-strain (C57BL/6 H-2b) antigen-presenting cells in the presence of interferon-γ. All Treg populations were examined phenotypically for expression of FoxP3 and evaluated for their ability to control rejection of fully MHC-mismatched heterotopic cardiac allografts. Treg were given on day -1 i.v. together with co-stimulation blockade deliberately designed to be sub-optimal (CTLA-4 Ig, 25μg day 0, 1, 5), in order to reveal any Treg-dependent impact on graft outcome. Results: Untreated CBA recipients rejected C57BL/6 hearts acutely (median survival time, MST, 8 days n=10). Sub-optimal CTLA-4 Ig treatment prolonged graft survival to an MST of 50 days, (n=8). When given in combination with sub-optimal CTLA-4 Ig, neither 2×106 freshly isolated nTreg (90-95% FoxP3+), nor 2×106 expanded-nTreg (90% FoxP3+) prolonged graft survival long-term and in fact, freshly isolated nTreg appeared to partially over-ride the effect of CTLA-4 Ig (MST=24 days n=8; MST=40 days n=5, fresh and expanded nTreg respectively). In clear contrast, delivery of 2×106 IFN-γ Treg led to a median survival time of 95 days (n=6), with 50% graft survival >100 days. The effect was dose-dependent (MST=66 days n=4; MST=100 days n=3, 0.5×106 and 4×106 IFN-γ Treg, respectively) and occurred despite the fact that this non-selected population was only between 40-50% FoxP3+. Furthermore, preliminary results indicate that the optimal time of IFN-γ Treg delivery is before rather than after transplant (MST 100 days, n=3 vs MST 18 days, n=4; delivery on day -3 vs. day +3 respectively) suggesting that early homing to the graft may be critical determinant in Treg efficacy. Conclusions: Although initial attempts to utilise Treg cell therapy in clinical transplantation will focus predominantly on expanded nTreg, our data indicate that efforts to develop alloantigen-driven adaptive Treg should not be overlooked. Whilst the generation of alloantigen-driven Treg will be challenging in terms of logistics, the data suggest that such cells may provide the most effective populations for cellular therapy and may offer a route to drug-minimisation without compromising transplant outcome.

Combined Therapy of Nrp1+ T Cells with Low-Dose Sirolimus Prolongs Fully MHC-Mismatched Cardiac Allograft Survival in Mice

Background: Neuropilin-1 (Nrp1), originally identified for its role in neuronal development, has recently been implicated in the regulation of CD4 T-cell immune responses in experimental autoimmune encephalitis. However, whether Nrp1 plays a role in T cell mediated allograft rejection remains unclear. The aim of this study was to investigate the regulatory effects of Nrp1+ T cells on allogeneic T lymphocyte proliferation in vitro, and whether combined therapy of Nrp1+ T cells with low-dose sirolimus could prolong the survival of fully MHC-mismatched cardiac allograft in mice. Methods: Nrp1+ T cells were sorted from C57BL/6J (BL/6) mice by flow cytometry, while CD4+CD25+ Treg cells were isolated from BL/6 mice using MACS magnetic beads. Nrp1+ T cells or CD4+CD25+ Treg cells were co-cultured with responder splenocytes from BL/6 mice stimulated with irradiated stimulator splenocytes from BALB/c mice. Proliferation was assessed by 3H-thymidine incorporation during the last 18 h of the 3-day culture. Intra-abdominal heterotopic heart transplant was performed from BALB/c mice to BL/6 mice. 2 × 106 Nrp1+ T cells or CD4+CD25+ Treg cells from BL/6 mice were adoptively transferred via tail vein into recipient mice weekly for two months. Sirolimus treatment was given daily i.p. (1 mg/kg/day) between d1 and d14 post transplantation. BL/6 recipients treated with PBS or sirolimus or Nrp1+ T cells were served as controls. Results: Nrp1+ T cells markedly suppressed the proliferation of allogeneic T lymphocyte (P < 0.01), with a suppressive property as potent as CD4+CD25+ Treg cells (P > 0.05). The survival of allografts treated with both Nrp1+ T cells and sirolimus was prolonged significantly as compared with PBS-treated allografts (77 ± 4 days versus 6 ± 2 days, P < 0.001), slightly less better than those treated with CD4+CD25+ Treg cells together with sirolimus (82 ± 5 days, P < 0.05). The survival of donor hearts in sirolimus control group and Nrp1+ T cells control group were prolonged to 21 ± 2 and 32 ± 3 days, respectively. Conclusions: Nrp1+ T cell served as a potent suppressor for allogeneic T lymphocyte proliferation. Adoptively transferred Nrp1+T cells in combination with low-dose sirolimus were effective in prolonging acute cardiac allograft survival in a robust murine model.