Sustained, indolent immune injury of the vasculature of a heart transplant limits long-term graft and recipient survival. This injury is mitigated by a poorly characterized, maladaptive repair response. Vascular endothelial cells respond to proangiogenic cues in the embryo by differentiation to specialized phenotypes, associated with expression of apelin. In the adult, the role of developmental proangiogenic cues in repair of the established vasculature is largely unknown. We found that human and minor histocompatibility–mismatched donor mouse heart allografts with alloimmune-mediated vasculopathy upregulated expression of apelin in arteries and myocardial microvessels. In vivo, loss of donor heart expression of apelin facilitated graft immune cell infiltration, blunted vascular repair, and worsened occlusive vasculopathy in mice. In vitro, an apelin receptor agonist analog elicited endothelial nitric oxide synthase activation to promote endothelial monolayer wound repair and reduce immune cell adhesion. Thus, apelin acted as an autocrine growth cue to sustain vascular repair and mitigate the effects of immune injury. Treatment with an apelin receptor agonist after vasculopathy was established markedly reduced progression of arterial occlusion in mice. Together, these initial data identify proangiogenic apelin as a key mediator of coronary vascular repair and a pharmacotherapeutic target for immune-mediated injury of the coronary vasculature.

Recovery of the T lymphocyte compartment within a lymphopenic host by lymphopenia induced proliferation (LIP) is regulated by inter- and intraclonal competition for limited resources including homeostatic cytokines and peptide:MHC (pMHC) complexes with which the TCR can interact at least weakly to yield a tonic signal. Importantly, the process of LIP can synergize with other factors that promote T cell activation to drive inflammatory disease. While reconstitution of the lymphoid compartment of immune deficient Rag-/- mice by transfer of wild-type hematopoietic stem cells (HSC) does not generally result in an overt disease phenotype, transfer of HSC deficient in expression of the co-inhibitory molecule PD-1 results in severe systemic autoimmunity driven by newly generated T cells that emerge from the thymus into the periphery and undergo LIP. Importantly, autoimmunity does not appear to depend on a response to exogenous (i.e. gut flora-derived) antigens. PD-1 is well known to be up-regulated during T cell activation in response to cognate antigens, but it is unclear whether PD-1 has a role in controlling LIP of T cells in the absence of cognate antigen, i.e. in response to tonic pMHC. We examined whether PD-1 controls LIP of newly generated T cells by controlling the response to tonic pMHC or the homeostatic cytokine IL-7. We found that PD-1 deficient T cells have a proliferative advantage over WT T cells during LIP and this effect is MHC-II dependent and independent of IL-7Rα signaling. Furthermore, our data suggest that signals through IL-7Rα can be dispensable for LIP and may instead be of increased importance for T cell survival in conditions of high competition for limited pMHC (e.g. post-LIP, in a lymphoreplete host). We hypothesize that autoimmunity post-PD-1-/- HSC transplant is the result of an overzealous T cell response to normally tonic self-pMHC precipitated by the synergy of LIP and PD-1 deficiency. Furthermore, potentiation of TCR signals in response to normally tonic self-pMHC may contribute to the success of PD-1 blockade in cancer immunotherapy.

Lymphopenia can result from various factors including viral infections, clinical interventions, or as a normal property of the fetal/neonatal period. T cells in a lymphopenic environment undergo lymphopenia-induced proliferation (LIP) to fill the available “niche” as defined by peptide-MHC (pMHC) and homeostatic cytokine resources. We recently reported systemic autoimmunity following reconstitution of the lymphoid compartment of Rag1-/- mice with PD-1-/- hematopoietic stem cells or by transfer of thymocytes, but not splenocytes, suggesting that PD-1 plays a crucial role in controlling recent thymic emigrants (RTE) and preventing autoimmunity upon their LIP. However, it is unclear whether RTE residing within the periphery of a lymphoreplete host maintain enhanced autoimmune generating potential or if this property only manifests if RTE experience a lymphopenic periphery immediately after export from the thymus. Furthermore, it is unclear which of a variety of T cell effector mechanisms generate pathology when control of RTE by PD-1 is lacking. Herein, we determined that PD-1 is upregulated on CD4 T cells undergoing the natural LIP characteristic of the neonatal period. Newly generated T cells lacking PD-1 maintained an enhanced autoimmune potential even after residence in a lymphoreplete periphery, emphasizing the importance of PD-1 in the establishment of peripheral tolerance. Neither Fas nor perforin-dependent killing mechanisms were required for autoimmunity, while host MHC-II expression was critical, suggesting that LIP-driven autoimmunity in the absence of PD-1 may primarily result from a CD4 T cell-mediated systemic cytokinemia, a feature potentially shared by other autoimmune or inflammatory syndromes associated with immune reconstitution and LIP.

Mixed hematopoietic chimerism induction as a way to foster tolerance to donor organs in recipients that have been sensitized to donor antigens is challenging. Donor specific antibodies (DSA) are a dominant barrier towards successful donor bone marrow engraftment. Although desensitization methods are routinely used in recipients with allo‐sensitization for allogeneic bone marrow transplantation, engraftment is frequently unsuccessful. To overcome the barrier of prior sensitization we tested enzymatic desensitization of donor‐specific IgG using imlifidase and EndoS, which both partially block the function of DSA in mice, as a novel approach to improve murine bone marrow engraftment in primed hosts. We found that EndoS was capable of inhibiting antibody‐mediated killing of donor cells in vivo. Furthermore, the effect of EndoS depended on the titer of DSA and the genetic background of the recipients. In combination with imlifidase, EndoS improved the survival of donor bone marrow cells. Together with cyclophosphamide, bortezomib, T cell depletion and non‐lethal irradiation, imlifidase in combination with EndoS allowed allogeneic bone marrow engraftment in sensitized recipients. We conclude that enzymatic inactivation of DSA, using the combination of imlifidase and EndoS can be used for inducing donor hematopoietic chimerism in allo‐sensitized recipient mice in combination with other desensitization strategies.

One strategy to prevent islet rejection is to create a favorable immune-protective local environment at the transplant site. Herein, we utilize localized cyclosporine A (CsA) delivery to islet grafts via poly(lactic-co-glycolic acid) (PLGA) microparticles to attenuate allograft rejection. CsA-eluting PLGA microparticles were prepared using a single emulsion (oil-in-water) solvent evaporation technique. CsA microparticles alone significantly delayed islet allograft rejection compared to islets alone (p < 0.05). Over 50% (6/11) of recipients receiving CsA microparticles and short-term cytotoxic T lymphocyte-associated antigen 4-Ig (CTLA4-Ig) therapy displayed prolonged allograft survival for 214 days, compared to 25% (2/8) receiving CTLA4-Ig alone. CsA microparticles alone and CsA microparticles + CTLA4-Ig islet allografts exhibited reduced T-cell (CD4⁺ and CD8⁺ cells, p < 0.001) and macrophage (CD68⁺ cells, p < 0.001) infiltration compared to islets alone. We observed the reduced mRNA expression of proinflammatory cytokines (IL-6, IL-10, INF-γ, and TNF-α; p < 0.05) and chemokines (CCL2, CCL5, CCL22, and CXCL10; p < 0.05) in CsA microparticles + CTLA4-Ig allografts compared to islets alone. Long-term islet allografts contained insulin⁺ and intra-graft FoxP3⁺ T regulatory cells. The rapid rejection of third-party skin grafts (C3H) in islet allograft recipients suggests that CsA microparticles + CTLA4-Ig therapy induced operational tolerance. This study demonstrates that localized CsA drug delivery plus short-course systemic immunosuppression promotes an immune protective transplant niche for allogeneic islets.

Heart transplant and recipient survival are limited by immune cell-mediated injury of the graft vasculature. We examined the role of the phosphoinositide 3-kinase-β (PI3Kβ) isoform in endothelial cells (EC) during coronary vascular immune injury and repair in mice. In minor histocompatibility-antigen mismatched allogeneic heart grafts, a robust immune response was mounted to each wild-type, PI3Kβ inhibitor-treated, or endothelial-selective PI3Kβ knockout (ECβKO) graft transplanted to wild-type recipients. However, microvascular EC loss and progressive occlusive vasculopathy only developed in control, but not PI3Kβ-inactivated hearts. We observed a delay in inflammatory cell infiltration of the ECβKO grafts, particularly in the coronary arteries. Surprisingly, this was accompanied by an impaired display of proinflammatory chemokine and adhesion molecules by the ECβKO ECs. In vitro, tumor necrosis factor α-stimulated endothelial ICAM1 and VCAM1 expression was blocked by PI3Kβ inhibition or RNA interference. Selective PI3Kβ inhibition also blocked tumor necrosis factor α-stimulated degradation of inhibitor of nuclear factor kappa Bα and nuclear translocation of nuclear factor kappa B p65 in EC. These data identify PI3Kβ as a therapeutic target to reduce vascular inflammation and injury.