Alarmins and Their Receptors as Modulators and Indicators of Alloimmune Responses.

Cell damage and death releases alarmins, self-derived immunomodulatory molecules that recruit and activate the immune system. Unfortunately, numerous processes critical to the transplantation of allogeneic materials result in the destruction of donor and recipient cells and may trigger alarmin release. Alarmins, often described as damage-associated molecular patterns, together with exogenous pathogen-associated molecular patterns, are potent orchestrators of immune responses; however, the precise role that alarmins play in alloimmune responses remains relatively undefined. We examined evolving concepts regarding how alarmins affect solid organ and allogeneic hematopoietic cell transplantation outcomes and the mechanisms by which self molecules are released. We describe how, once released, alarmins may act alone or in conjunction with nonself materials to contribute to cytokine networks controlling alloimmune responses and their intensity. It is becoming recognized that this class of molecules has pleotropic functions, and certain alarmins can promote both inflammatory and regulatory responses in transplant models. Emerging evidence indicates that alarmins and their receptors may be promising transplantation biomarkers. Developing the therapeutic ability to support alarmin regulatory mechanisms and the predictive value of alarmin pathway biomarkers for early intervention may provide opportunities to benefit graft recipients.

In Vivo Mobilization and Functional Characterization of Nonhuman Primate Monocytic Myeloid-Derived Suppressor Cells.

Increasing evidence from small animal models shows that myeloid-derived suppressor cells (MDSCs) can play a crucial role in inhibiting allograft rejection and promoting transplant tolerance. We identified CD3(-)CD20(-)HLA-DR(-)CD14(+)CD33(+)CD11b(+) cells in peripheral blood of healthy rhesus macaques. These putative monocytic MDSCs constituted 2.1% ± 1.7% of lin(-)HLA-DR(-) peripheral blood mononuclear cells. Administration of granulocyte-macrophage colony-stimulating factor (CSF) and granulocyte CSF increased their incidence to 5.3% ± 3.4%. The total number of MDSCs that could be flow sorted from a single whole rhesus leukapheresis product was 38 ± 13 × 10(6) (n = 10 monkeys). Freshly isolated or cryopreserved MDSCs from mobilized monkeys incorporated in cultures of anti-CD3- and anti-CD28-stimulated autologous T cells markedly suppressed CD4(+) and CD8(+) T cell proliferation and cytokine secretion (interferon γ, IL-17A). Moreover, these MDSCs enhanced CD4(+)CD25(hi)Foxp3(+) regulatory T cell (Treg) expansion while inhibiting proliferation of activated memory T cells and increasing Treg relative to effector and terminally differentiated memory T cells. Inhibition of arginase-1, but not inducible nitric oxide synthase activity, partially reversed the inhibitory effect of the MDSCs on CD8(+) T cell proliferation. Consequently, functional MDSCs can be isolated from nonhuman primates for prospective use as therapeutic cellular vaccines in transplantation.

Elevated ST2 Distinguishes Incidences of Pediatric Heart and Small Bowel Transplant Rejection.

Elevated serum soluble (s) suppressor of tumorigenicity-2 is observed during cardiovascular and inflammatory bowel diseases. To ascertain whether modulated ST2 levels signify heart (HTx) or small bowel transplant (SBTx) rejection, we quantified sST2 in serially obtained pediatric HTx (n = 41) and SBTx recipient (n = 18) sera. At times of biopsy-diagnosed HTx rejection (cellular and/or antibody-mediated), serum sST2 was elevated compared to rejection-free time points (1714 ± 329 vs. 546.5 ± 141.6 pg/mL; p = 0.0002). SBTx recipients also displayed increased serum sST2 during incidences of rejection (7536 ± 1561 vs. 2662 ± 543.8 pg/mL; p = 0.0347). Receiver operator characteristic (ROC) analysis showed that serum sST2 > 600 pg/mL could discriminate time points of HTx rejection and nonrejection (area under the curve [AUC] = 0.724 ± 0.053; p = 0.0003). ROC analysis of SBTx measures revealed a similar discriminative capacity (AUC = 0.6921 ± 0.0820; p = 0.0349). Quantitative evaluation of both HTx and SBTx biopsies revealed that rejection significantly increased allograft ST2 expression. Pathway and Network Analysis of biopsy data pinpointed ST2 in the dominant pathway modulated by rejection and predicted tumor necrosis factor-α and IL-1ß as upstream activators. In total, our data indicate that alloimmune-associated pro-inflammatory cytokines increase ST2 during rejection. They also demonstrate that routine serum sST2 quantification, potentially combined with other biomarkers, should be investigated further to aid in the noninvasive diagnosis of rejection.

Adenosine triphosphate-competitive mTOR inhibitors: a new class of immunosuppressive agents that inhibit allograft rejection.

The mechanistic/mammalian target of rapamycin (mTOR) is inhibited clinically to suppress T cell function and prevent allograft rejection. mTOR is the kinase subunit of two mTOR-containing complexes, mTOR complex (mTORC) 1 and 2. Although mTORC1 is inhibited by the macrolide immunosuppressant rapamycin (RAPA), its efficacy may be limited by its inability to block mTORC1 completely and its limited effect on mTORC2. Adenosine triphosphate (ATP)-competitive mTOR inhibitors are an emerging class of mTOR inhibitors that compete with ATP at the mTOR active site and inhibit any mTOR-containing complex. Since this class of compounds has not been investigated for their immunosuppressive potential, our goal was to determine the influence of a prototypic ATP-competitive mTOR inhibitor on allograft survival. AZD8055 proved to be a potent suppressor of T cell proliferation. Moreover, a short, 10-day course of the agent successfully prolonged murine MHC-mismatched, vascularized heart transplant survival. This therapeutic effect was associated with increased graft-infiltrating regulatory T cells and reduced CD4(+) and CD8(+) T cell interferon-γ production. These studies establish for the first time, that ATP-competitive mTOR inhibition can prolong organ allograft survival and warrant further investigation of this next generation mTOR inhibitors.

Rapamycin augments human DC IL-12p70 and IL-27 secretion to promote allogeneic Type 1 polarization modulated by NK cells.

Mammalian target of rapamycin kinase inhibitor (mTORi) rapamycin (RAPA) use in transplantation can lead to inflammatory complications in some patients. Our goal was to better understand how mTORi-exposed human monocyte-derived dendritic cells (DC) stimulated with pro-inflammatory cytokines shape T cell allo-immunity. RAPA-conditioned-DC (RAPA-DC) displayed a more immature phenotype than untreated, control (CTRL)-DC. However, subsequent exposure of RAPA-DC to an inflammatory cytokine cocktail (ICC) plus IFN-γ induced a mature Type-1 promoting phenotype, consisting of elevated HLA-DR and co-stimulatory molecules, augmented IL-12p70 and IL-27 production, but decreased IL-10 secretion compared to CTRL-DC. Co-culture of mature (m)RAPA-DC with allogeneic peripheral blood mononuclear cells resulted in significantly increased Type-1 (IFN-γ) responses by T cells. Moreover, NK cells acted as innate modulators that conveyed activating cell-to-cell contact signals in addition to helper (IFN-γ) and/or regulatory (IL-10) soluble cytokines. We conclude that production of IL12-p70, IL-27 and low IL-10 by RAPA-DC allowed us to elucidate how these cytokines as well as NK-DC interaction shapes T cell allo-immunity. Thus, lack of inhibitory NK cell function during allo-specific T cell activation by human ICC + IFN-γ-stimulated RAPA-DC may represent an unwanted effector mechanism that may underlie RAPA-induced inflammatory events in transplant patients undergoing microbial infection or allograft rejection.