Silk fibroin preserves beta cell function under inflammatory stress while stimulating islet cell surface GLUT2 expression.

Silk fibroin is a novel biomaterial for enhancing transplanted islet cell function and survival. This study investigated whether silk fibroin may have unique properties that improve islet function in the face of inflammatory-mediated stress during transplantation. Murine islet function was tested in vitro with either silk fibroin or alginate and challenged with inflammatory cytokines. The glucose-stimulated insulin secretion index for all conditions decreased with inflammatory cytokines, but was better preserved for islets exposed to silk compared to those exposed to alginate or medium. GLUT2 transporter expression on the cell surface of islets exposed to silk was increased compared to alginate or medium alone. Upon cytokine stress, a greater percentage of islet cells exposed to silk expressed GLUT2 on their surface. We conclude that preconditioning islets with silk fibroin stimulates islet cell surface GLUT2 expression, an increase, which persists under inflammatory stress, and may improve islet engraftment and function after transplantation.

What's hot, what's new at WTC--basic science.

The World Transplant Congress of 2014 presented a broad swath of science that touched on many disparate aspects of cell and organ transplantation, molecular and cellular immunology, systems biology, development, technology and translation into humans. A number of themes emerged this year. B cell biology and antibody chemistry were prominent, as they have been for several years. T cells, co-stimulatory blockade and regulatory T cells continue to dominate many aspects of immune research. Many new aspects of monocyte, macrophage, NK cell and NK T cell development, biology and regulation are now being explored. Diverse aspects of organ injury and the acute and chronic responses to injury are being investigated with new techniques, new targets and a resurgent vigor. Novel advances in xenotransplantation and experimental tolerance garnered much attention. Newer investigations in microbiota and nanotechnology promise significant gains in the near future. Lastly the 'omics of DNA, RNA, proteins, metabolites, bacteria and enzyme actions promise new understanding in biological systems and how to control those systems.

Human microbiota characterization in the course of renal transplantation.

Recent studies demonstrate that the human microbiota, the collection of microorganisms growing on and in individuals, have numerous bidirectional interactions with the host, influencing immunity, resistance to infection, inflammation and metabolism. Little has been done to study the potential associations between microbiota composition and transplant outcome. Here, we investigated the longitudinal changes in the blood, urinary, oral and rectal microbiota of renal allograft recipients before and at 1 and 6 months after transplantation. The results showed major changes in microbiota composition as a result of the transplant episode and associated medications, and these changes persisted over time. The high interindividual variation as well as differences in response to transplantation suggested that it is unlikely that the same specific microbiota members can serve as universal diagnostic markers. Rather, longitudinal changes in each individual's microbiota have the potential to be indicative of health or disease. Use of sensitive nucleic acid-based testing showed that urine, irrespective of disease states, more often harbors a diverse microbiota than appreciated by conventional culture techniques. These results lay the groundwork to construct more comprehensive future investigations to identify microbiota characteristics that can serve as diagnostic markers for transplant health and to guide intervention strategies to improve transplant outcome.

Lymphotoxin-beta receptor blockade induces inflammation and fibrosis in tolerized cardiac allografts.

The lymphotoxin system (LT) regulates interactions between lymphocytes and stromal cells to maintain lymphoid microenvironmental homeostasis. Soluble LT beta-receptor-Ig (LTßRIg) blocks lymphocyte LTα1ß2-stromal cell LTßR signaling. In a murine cardiac allograft model, LTbRIg treatment reversed the tolerance induced by anti-CD40L antibody leading to graft inflammation and fibrosis. LTßRIg treatment decreased PD-L1 expression by blood endothelial cells, and decreased VCAM-1 while increasing CXCL1, CXCL2, CXCL12, CCL5, CCL21 and IL-6 expression in fibroblastic reticular cells. In secondary lymphoid organs these effects caused T- and B cell zone disruption, loss of CD35(+) follicular dendritic cells and abnormal recruitment of CD11b(+) Ly6G(+) neutrophils. These disruptions correlated with increased numbers of CD8(+) T cells and CD11b(+) Ly6G(+) neutrophils, and decreased numbers of CD4(+) T cells and Foxp3(+) regulatory T cells in the grafts. Depleting neutrophils or blocking neutrophil-attracting chemokines restored normal histology in lymph node, spleen and grafts. Taken together, LTßRIg treatment altered stromal subset, particularly fibroblastic reticular cell, production of cytokines and chemokines, resulting in changes in neutrophil recruitment in spleen, lymph node and grafts, and inflammation and fibrosis associated with decreased Foxp3(+) regulatory T cells and increased CD8(+) T cell infiltration of grafts.

Fates of CD4+ T cells in a tolerant environment depend on timing and place of antigen exposure.

In experimental organ transplantation, tolerance is induced by administration of anti-CD40L mAb in conjunction with donor-specific splenocyte transfusion. Multiple, sometimes conflicting mechanisms of action resulting from this treatment have been reported. To resolve these issues, this study assessed the fates of graft reactive cells at different times and locations in the tolerant environment. Alloantigen-specific CD4(+) T cells transferred at time of tolerance induction (7 days before transplantation) became activated, expressed CD69 and CD44, and proliferated. Importantly, a large subset of this population became Foxp3(+) , more so in the lymph nodes than spleen, indicative of differentiation to a regulatory phenotype. In contrast, graft reactive CD4(+) T cells transferred to tolerogen-treated recipients at the time of transplantation failed either to proliferate or to differentiate, and instead were deleted via apoptosis. In untreated rejecting recipients graft reactive CD4(+) T cells became activated, proliferated and differentiated mainly in the spleen, and many of these cells were eventually deleted. These data resolve many apparent contradictions in the literature by showing that the timing of antigen exposure, the immunologic status of the recipients and secondary lymphoid organ location act together as key factors to determine the fate of graft reactive CD4(+) T cells.

Distinct inflammatory signals have physiologically divergent effects on epigenetic regulation of Foxp3 expression and Treg function.

Foxp3 expression in regulatory T cells (Treg) is required for their development and suppressive function. How different inflammatory signals affect Foxp3 chromatin structure, expression and Tregs plasticity are not completely known. In the present study, the Toll-like receptor 2 (TLR2) ligand peptidoglycan inhibited Foxp3 expression in both natural Treg (nTreg) and TGFß-driven adaptive Treg (aTreg). Inhibition was independent of paracrine Th1, Th2 and Th17 cytokines. PGN-induced T cell-intrinsic TLR2-Myd88-dependent IFR1 expression and induced IRF1 bound to IRF1 response elements (IRF-E) in the Foxp3 promoter and intronic enhancers, and negatively regulated Foxp3 expression. Inflammatory IL-6 and TLR2 signals induced divergent chromatin changes at the Foxp3 locus and regulated Treg suppressor function, and in an islet transplant model resulted in differences in their ability to prolong graft survival. These findings are important for understanding how different inflammatory signals can affect the transplantation tolerance and immunity.

Guidelines for the diagnosis of antibody-mediated rejection in pancreas allografts-updated Banff grading schema.

The first Banff proposal for the diagnosis of pancreas rejection (Am J Transplant 2008; 8: 237) dealt primarily with the diagnosis of acute T-cell-mediated rejection (ACMR), while only tentatively addressing issues pertaining to antibody-mediated rejection (AMR). This document presents comprehensive guidelines for the diagnosis of AMR, first proposed at the 10th Banff Conference on Allograft Pathology and refined by a broad-based multidisciplinary panel. Pancreatic AMR is best identified by a combination of serological and immunohistopathological findings consisting of (i) identification of circulating donor-specific antibodies, and histopathological data including (ii) morphological evidence of microvascular tissue injury and (iii) C4d staining in interacinar capillaries. Acute AMR is diagnosed conclusively if these three elements are present, whereas a diagnosis of suspicious for AMR is rendered if only two elements are identified. The identification of only one diagnostic element is not sufficient for the diagnosis of AMR but should prompt heightened clinical vigilance. AMR and ACMR may coexist, and should be recognized and graded independently. This proposal is based on our current knowledge of the pathogenesis of pancreas rejection and currently available tools for diagnosis. A systematized clinicopathological approach to AMR is essential for the development and assessment of much needed therapeutic interventions.

Mechanisms of regulation of cell-mediated immunity. VII. Suppressor T cells induced by suboptimal doses of antigen plus an I-J-specific allogeneic effect.

Intravenous injection of 0.01 mM 2,4,6-trinitrobenzene sulfonic acid-derivatized syngeneic lymphoid cells generates a Thy-1-positive, antigen-specific suppressor cell for contact sensitivity which requires an I-J allogeneic effect to become fully activated. It is necessary and sufficient for all allogeneic effect to be directed solely against the suppressor cell, and once activated, the cell can suppress in an H-2-unrestricted fashion. The results are discussed in the framework of entry into the suppressor pathway, the allogeneic effect as a reflection of normal physiologic processes, and the importance of I-J as a receptor and signal among cells in the suppressor pathway.

A single amino acid determines the immunostimulatory activity of interleukin 10.

Cellular interleukin 10s (cIL-10s) of human and murine origin have extensive sequence and structural homology to the Epstein-Barr virus BCRF-I gene product, known as viral IL-10 (vIL-10). Although these cytokines share many immunosuppressive properties, vIL-10 lacks several of the immunostimulatory activities of cIL-10 on certain cell types. The molecular and cellular bases for this dichotomy are not currently defined. Here, we show that the single amino acid isoleucine at position 87 of cIL-10 is required for its immunostimulatory function. Substitution of isoleucine in cIL-10 with alanine, which corresponds to the vIL-10 residue, abrogates immunostimulatory activity for thymocytes, mast cells, and alloantigenic responses while preserving immunosuppressive activity for inhibition of interferon gamma production and prolongation of cardiac allograft survival. Conversely, substitution of alanine with isoleucine in vIL-10 converts it to a cIL-10-like molecule with immunostimulatory activity. This single conservative residue alteration significantly affects ligand affinity for receptor; however, affinity changes do not necessarily alter specific activities for biologic responses in a predictable fashion. These results suggest complex regulation of IL-10 receptor-ligand interactions and subsequent biological responses. These results demonstrate that vIL-10 may represent a captured and selectively mutated cIL-10 gene that benefits viral pathogenesis by leading to ineffective host immune responses. The ability to manipulate the activity of IL-10 in either a stimulatory or suppressive direction may be of practical value for regulating immune responses for disease therapy, and of theoretical value for determining what aspects of IL-10 activity are important for normal T cell responses.

Anti-CD2 receptor and anti-CD2 ligand (CD48) antibodies synergize to prolong allograft survival.

Indefinite graft survival was obtained with murine cardiac allografts using the combined administration of monoclonal antibodies (mAbs) directed against the receptor ligand pair CD2-CD48. Although each antibody could prolong graft survival when given alone, neither resulted in the indefinite graft survival seen with the combination. Combined mAb administration is associated with inhibition of T cell priming and help and subsequent cytotoxic T lymphocyte generation. This indicates that the interaction between CD2 and its ligand is important for antigen priming and recognition, and combined mAbs may prove to be a useful therapeutic regimen for transplantation.

Natural killer cells and the immune response in solid organ transplantation.

Natural killer (NK) cells have been characterized classically for their cytotoxicity against pathogen infected or stressed cells as well as for their role in monitoring the expression of self MHC I. However, the participation of NK cells in solid organ transplantation (SOT) is poorly defined due to conflicting clinical and animal model data. Preclinical models have shown that NK cells exacerbate T-cell allogeneic responses during rejection, but can also promote tolerance induction under immunosuppressive conditions. Further, while protocols such as costimulatory blockade effectively induce tolerance by blocking T-cell activation and promoting Treg generation, how such regimens regulate other innate and adaptive immune cells, including NK cells, is incomplete. This review examines NK cells and the regulation of their effector functions in SOT.

Evolving paradigms that determine the fate of an allograft.

Despite the many advances in both immunological knowledge and the practical application of clinical immunosuppression, the holy grail of indefinite graft survival with immune tolerance in clinical solid organ transplantation remains a distant dream. The tremendous progress made in understanding the molecular and cellular basis of allograft rejection has not been translated into durable modalities that have advanced clinical care and outcomes. Indeed, currently used drugs and treatment protocols, largely directed at inhibiting alloreactive T cells, have not optimally improved allograft survival or function. A shift in emphasis, focusing on under appreciated immune pathways must now be considered to make further improvement. We highlight three areas of recent interest, complement, NK cells and lymphatics, which reinforce the concept that the transplant community must direct attention on how the immune system as a whole responds to a transplant. The current challenge is to integrate molecular, cellular and anatomic concepts to achieve the equivalent of a unified field theory of the immune response to organ transplants.

Immune reconstitution following rabbit antithymocyte globulin.

Depletional induction therapies are routinely used to prevent acute rejection and improve transplant outcome. The effects of depleting agents on T-cell subsets and subsequent T-cell reconstitution are incompletely defined. We used flow cytometry to examine the effects of rabbit antithymocyte globulin (rATG) on the peripheral T-cell repertoire of pediatric and adult renal transplant recipients. We found that while rATG effectively depleted CD45RA+CD27+ naïve and CD45RO+CD27+ central memory CD4+ T cells, it had little effect on CD45RO+CD27- CD4+ effector memory or CD45RA+CD31-, CD45RO+CD27+ and CD45RO+CD27- CD8+ T cell subsets. When we performed a kinetic analysis of CD31+ recent thymic emigrants and CD45RA+/RO+ T cells, we found evidence for both thymopoiesis and homeostatic proliferation contributing to immune reconstitution. We additionally examined the impact of rATG on peripheral CD4+Foxp3+ T cells. We found that in adults, administration of rATG-induced peripheral expansion and new thymic emigration of T cells with a Treg phenotype, while CD4+Foxp3+ T cells of thymic origin predominated in children, providing the first evidence that rATG induces Treg in vivo. Collectively our data indicate that rATG alters the balance of regulatory to memory effector T cells posttransplant, providing an explanation for how it positively impacts transplant outcome.

Human FOXP3+ regulatory T cells in transplantation.

CD4+CD25+FOXP3+ suppressive regulatory T cells (Treg) represent a subset of immune regulatory cells. Based on experimental results, Treg have recently been considered as a potential treatment option in several diseases. Compared with murine Treg, human CD4+CD25+FOXP3+ cells are less well characterized and understood, so a thorough understanding of their biology is vital before clinical applications can be initiated. This review summarizes knowledge on generation, phenotypic characteristics and function of human Treg. The possible role of these cells in organ transplantation, as well as interactions between immunosuppression and Treg are also discussed.

Calcineurin-inhibitor-free immunosuppression based on the JAK inhibitor CP-690,550: a pilot study in de novo kidney allograft recipients.

This randomized, pilot study compared the Janus kinase inhibitor CP-690,550 (15 mg BID [CP15] and 30 mg BID [CP30], n = 20 each) with tacrolimus (n = 21) in de novo kidney allograft recipients. Patients received an IL-2 receptor antagonist, concomitant mycophenolate mofetil (MMF) and corticosteroids. CP-690,550 doses were reduced after 6 months. Due to a high incidence of BK virus nephropathy (BKN) in CP30, MMF was discontinued in this group. The 6-month biopsy-proven acute rejection rates were 1 of 20, 4 of 20 and 1 of 21 for CP15, CP30 and tacrolimus groups, respectively. BKN developed in 4 of 20 patients in CP30 group. The 6-month rates of cytomegalovirus disease were 2 of 20, 4 of 20 and none of 21 for CP15, CP30 and tacrolimus groups, respectively. Estimated glomerular filtration rate was >70 mL/min at 6 and 12 months (all groups). NK cells were reduced by

IL-10 immunosuppression in transplantation.

The description of IL-10 as an immunosuppressive cytokine that inhibits numerous T-cell and antigen-presenting cell functions has led to the investigation of the role of IL-10 in graft acceptance and rejection, in both manipulated and unmanipulated experimental systems. Recent results suggest a complex interaction of IL-10 with other cytokines and numerous cell types. Although IL-10 is not a simple immunosuppressive molecule, manipulation of its actions may be a useful approach for prolonging graft survival and preventing rejection.

Inhibition of angiogenesis is associated with reduced islet engraftment in diabetic recipient mice.

Rapid reestablishment of a functional microvasculature in transplanted islets is crucial for islet survival and function. To illustrate the importance of angiogenesis in islet engraftment, we took a loss-of-function approach to block angiogenesis in newly transplanted islets and determined the extent of islet engraftment in correlation with islet mass and glycemic control in diabetic recipient mice. Diabetic mice were transplanted with a marginal mass of 200 islets under the renal capsule, followed by once-daily oral administration of saline or 150 mg/kg of C-statin, a potent angiogenic inhibitor, for 14 days. Blood glucose profiles and the amplitude of glucose-stimulated insulin secretion in engrafted islets were determined. At 30 days posttransplant, islet grafts were retrieved for the determination of insulin content and vascular density by immunohistochemistry. When compared to sham-treated controls, diabetic recipient mice receiving a daily oral dose of C-statin exhibited significantly impaired blood glucose profiles and diminished glucose-stimulated insulin secretion in response to glucose challenge, correlating with significantly reduced intragraft insulin content and vascular density. Selective inhibition of angiogenesis was associated with reduced islet mass in diabetic mice. These data extend our view that rapid onset of angiogenesis is crucial for islet survival and engraftment and support the development of therapeutic strategies to stimulate angiogenesis in newly implanted islets for enhancing islet engraftment and improving the outcome of marginal islet transplantation.

Gene transfer of transforming growth factor-beta 1 prolongs murine cardiac allograft survival by inhibiting cell-mediated immunity.

Delivery of immunosuppressants directly to allografts using gene transfer and gene therapy approaches may inhibit immune activation while avoiding the systemic toxicity of conventional immunosuppression. Cardiac grafts from allogeneic (C57BL/6, H-2b) donors were transplanted into CBA/J (H-2k) recipients in a heterotopic, non-vascularized model pSVTGF-beta 1, a plasmid encoding murine transforming growth factor-beta 1 (TGF-beta 1) under the control of an SV40 promoter, was directly injected into grafts at surgery and prolonged survival from 12.0 +/- 0.7 to 25.1 +/- 2.1 days (p < 0.001) in a dose-dependent manner. Plasmid gene transfer-induced immunosuppression was localized to the area of the graft because plasmid injected remote from the graft did not prolong allograft survival and systemic immunity was not influenced by local gene transfer. Limiting dilution analysis of graft-infiltrating cells demonstrated that gene transfer reduced the precursor frequency of donor-specific cytotoxic T lymphocytes (CTL) and activated and total interleukin-2 (IL-2) producing helper T lymphocytes (HTL) in graft-infiltrating cells, whereas CTL generation and HTL precursor frequency in splenic lymphocytes were not altered. Additional data revealed that gene transfer inhibited the priming of TH0 cells and the conversion of primed TH1 cells to activated cells without the participation of TH2 suppressors. These data demonstrate that gene transfer of plasmid DNA encoding TGF-beta 1 in vivo suppresses local T cell immunity, which prolongs allograft survival.