Basic sciences in development: What changes will we see in transplantation in the next five years?

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Intragraft Cytokine mRNA Expression After Clinical Organ Transplantation

As the knowledge of the cytokine network in experimental transplant models grows, we need to understand how and to what extent cytokines mediate the various donordirected immune events in clinical situations. This overview on clinical cytokine measurements shows that specific intragraft cytokine messengerRNA (mRNA) expression profiles can be associated with acute rejection, may reflect the efficacy of immunosuppression, and can trace patients at risk for the development of early chronic rejection. Moreover, the literature also showed that acute rejection and immunological quiescence in man are not restricted to the cytokine patterns according to the type 1/type 2 paradigm. This apparent lack of association may be caused by the immunosuppression used in the clinic but may also be the result of the infinite diversity of donor and recipient factors of which polymorphisms in cytokines and cytokine receptor genes may playa central role

Mesenchymal stromal cells for organ transplantation: Different sources and unique characteristics?

PURPOSE OF THE REVIEW: In this review, recent findings on the effects of tissue and donor origin, culturing conditions and preconditioning regimens on the therapeutic effect of mesenchymal stem cells (MSC) in organ transplantation are discussed and the importance of understanding the characteristics of MSC for developing efficient therapy is stressed. RECENT FINDINGS: MSC research in organ transplantation is currently moving from safety-feasibility studies to efficacy studies and finding the optimal MSC for therapy is therefore highly relevant. Although sharing basic properties, there are subtle differences between MSC from different tissue sources that may affect their efficacy. Furthermore, the use of MSC from diseased organ recipients, donor or third party may affect their therapeutic effect. The importance of these differences in MSC properties may however be overshadowed by the impact of culture conditions on MSC. Culture conditions dramatically change the characteristics of MSC, and this situation can be exploited by exposing MSC to preconditioning treatment to bring about the desired properties in MSC. As MSC appear to be short-lived after infusion, the specific characteristics of MSC are mostly relevant for short-term interactions between MSC and host cells, which will subsequently take over the effects of MSC. The multiple effects of MSC are by no means unique, but the full spectrum of the effects in combination with their easy isolation and expansion make MSC a suitable cell type for therapy. SUMMARY: Tissue source, donor source and culture conditions affect the phenotypical and functional properties of MSC. The efficacy of MSC therapy will therefore depend on the source and manipulation of MSC

Toward development of imesenchymal stem cells for immunomodulatory therapy

Mesenchymal stem cells (MSC) are under development as an immunomodulatory therapy. The anticipated immunomodulatory effects of MSC are broad, from direct inhibition of lymphocyte proliferation, induction of regulatory T and B cells, to resetting the immune system via a hit-and-run principle. There are endless flavors of MSC. Differences between MSC are originating from donors variation, differences in tissue of origin, the effects of culture conditions, and expansion time. Even standard culture conditions change the properties of MSC dramatically and generate MSC that only remotely resemble their in vivo counterparts. Adjustments in culture protocols can further emphasize properties of interest in MSC, thereby generating cells fitted for specific purposes. Culture improved immunomodulatory MSC can be designed to target particular immune disorders. In this review, we describe the observed and the desired immunomodulatory effects of MSC and propose approaches how MSC with optimal immunomodulatory properties can be developed

The role of follicular T helper cells in the humoral alloimmune response after clinical organ transplantation

Over the past decade, antibody-mediated or humoral rejection in combination with development of de novo donor-specific antibodies (DSA) has been recognized as a distinct and common cause of transplant dysfunction and is responsible for one-third of the failed allografts. Detailed knowledge of the mechanisms that initiate and maintain B-cell driven antidonor reactivity is required to prevent and better treat this antidonor response in organ transplant patients. Over the past few years, it became evident that this response largely depends on the actions of both T follicular helper (Tfh) cells and the controlling counterparts, the T follicular regulatory (Tfr) cells. In this overview paper, we review the latest insights on the functions of circulating (c)Tfh cells, their subsets Tfh1, Tfh2 and Tfh17 cells, IL-21 and Tfr cells in antibody mediated rejection (ABMR). This may offer new insights in the process to reduce de novo DSA secretion resulting in a decline in the incidence of ABMR. In addition, monitoring these cell populations could be helpful for the development of biomarkers identifying patients at risk for ABMR and provide novel therapeutic drug targets to treat ABMR

End-stage renal disease causes skewing in the TCR Vβ-repertoire primarily within CD8+ T Cell subsets

A broad T cell receptor (TCR-) repertoire is required for an effective immune response. TCR-repertoire diversity declines with age. End-stage renal disease (ESRD) patients have a prematurely aged T cell system which is associated with defective T cell-mediated immunity. Recently, we showed that ESRD may significantly skew the TCR Vβ-repertoire. Here, we assessed the impact of ESRD on the TCR Vβ-repertoire within different T cell subsets using a multiparameter flow-cytometry-based assay, controlling for effects of aging and CMV latency. Percentages of 24 different TCR Vβ-families were tested in circulating naive and memory T cell subsets of 10 ESRD patients and 10 age- and CMV-serostatus-matched healthy individuals (HI). The Gini-index, a parameter used in economics to describe the distribution of income, was calculated to determine the extent of skewing at the subset level taking into account frequencies of all 24 TCR Vβ-families. In addition, using HI as reference population, the differential impact of ESRD was assessed on clonal expansion at the level of an individual TCR Vβ-family. CD8+, but not CD4+, T cell differentiation was

Interferon-gamma DNA methylation is affected by mycophenolic acid but not by tacrolimus after T-cell activation

Immunosuppressive drug therapy is required to treat patients with autoimmune disease and patients who have undergone organ transplantation. The main targets of the immunosuppressive drugs tacrolimus and mycophenolic acid (MPA; the active metabolite of mycophenolate mofetil) are T cells. It is currently unknown whether these immunosuppressive drugs have an effect on DNA methylation-an epigenetic regulator of cellular function. Here, we determined the effect of tacrolimus and MPA on DNA methylation of the gene promoter region of interferon gamma (IFNγ), a pro-inflammatory cytokine. Total T cells, naive T cells (CCR7+CD45RO-), and memory T cells (CD45RO+ and CCR7-CD45RO-) were isolated from CMV seropositive healthy controls and stimulated with α-CD3/CD28 in the presence or absence of tacrolimus or MPA. DNA methylation of the IFNγ promoter region was quantified by pyrosequencing at 4 h, days 1, 3, and 4 after stimulation. In parallel, T-cell differentiation, and IFNγ protein production were analyzed by flow cytometry at days 1 and 3 after stimulation. Our results show that MPA induced changes in IFNγ DNA methylation of naive T cells; MPA counteracted the decrease in methylation after stimulation. Tacrolimus did not affect IFNγ DNA methylation of naive T cells. In the memory T cells, both immunosuppressive drugs did not affect IFNγ DNA methylation. Differentiation of naive T cells into a central-memory-like phenotype (CD45RO+) was inhibited by both immunosuppressive drugs, while differentiation of memory T cells remained unaffected by both MPA and tacrolimus. IFNγ protein production was suppressed by tacrolimus. Our results demonstrate that MPA influenced IFNγ DNA methylation of naive T cells after stimulation of T cells, while tacrolimus had no effect. Both tacrolimus and MPA did not affect IFNγ DNA methylation of memory T cells

Phospho-specific flow cytometry for pharmacodynamic monitoring of immunosuppressive therapy in transplantation

Organ transplant recipients frequently suffer from toxicity or from lack of efficacy of immunosuppressive drugs, which can be attributed to individual variations in drug sensitivity. This problem can be resolved by applying pharmacodynamic monitoring that focuses on measuring the biological effects of drugs. Here we discuss the new technique called phospho-specific flow cytometry to monitor the activity of intracellular immune signaling pathways at the single-cell level in whole blood samples. Through this tool the efficacy of immunosuppressive medication can be assessed, novel targets can be identified, and differences in drug sensitivity between cells and patients can be clarified

Down-regulation of surface CD28 under belatacept treatment: An escape mechanism for antigen-reactive T-cells

Background The co-stimulatory inhibitor of the CD28-CD80/86-pathway, belatacept, allows calcineurininhibitor-free immunosuppression in kidney transplantation. However, aggressive T-cell mediated allogeneic responses have been observed in belatacept-treated patients, which could be explained by effector-memory T-cells that lack membrane expression of CD28, i.e. CD28-negative (CD28NULL) T-cells. CD28-positive (CD28POS) T-cells that down regulate their surface CD28 after allogeneic stimulation could also pose a threat against the renal graft. The aim of this study was to investigate this potential escape mechanism for CD28POS T-cells under belatacept treatment. Materials & Methods PBMCs, isolated T-cell memory subsets and isolated CD28POS T-cells were obtained from end-stage renal disease (ESRD) patients and co-cultured with allo-antigen in the presence of belatacept to mimic allogeneic reactions in kidney-transplant patients under belatacept treatment. As a control, IgG was used in the absence of belatacept. Results Despite high in vitro belatacept concentrations, a residual T-cell growth of ±30% was observed compared to the IgG control after allogeneic stimulation. Of the alloreactive Tcells, the majority expressed an effector-memory phenotype. This predominance for effector-memory T-cells within the proliferated cells was even larger when a higher dose of belatacept was added. Contrary to isolated naïve and central-memory T cells, isolated effectormemory T cells could not be inhibited by belatacept in differentiation or allogeneic IFNγ production. The proportion of CD28-positive T cells was lower within the proliferated T cell population, but was still substantial. A fair number of the isolated initially CD28POS T-cells differentiated into CD28NULL T-cells, which made them not targetable by belatacept. These induced CD28NULL T-cells were not anergic as they produced high amounts of IFNγ upon allogeneic stimulat