IL-3 Triggers Chronic Rejection of Cardiac Allografts by Activation of Infiltrating Basophils.

Chronic rejection is a major problem in transplantation medicine, largely resistant to therapy, and poorly understood. We have shown previously that basophil-derived IL-4 contributes to fibrosis and vasculopathy in a model of heart transplantation with depletion of CD4+ T cells. However, it is unknown how basophils are activated in the allografts and whether they play a role when cyclosporin A (CsA) immunosuppression is applied. BALB/c donor hearts were heterotopically transplanted into fully MHC-mismatched C57BL/6 recipients and acute rejection was prevented by depletion of CD4+ T cells or treatment with CsA. We found that IL-3 is significantly upregulated in chronically rejecting allografts and is the major activator of basophils in allografts. Using IL-3-deficient mice and depletion of basophils, we show that IL-3 contributes to allograft fibrosis and organ failure in a basophil-dependent manner. Also, in the model of chronic rejection involving CsA, IL-3 and basophils substantially contribute to organ remodeling, despite the almost complete suppression of IL-4 by CsA. In this study, basophil-derived IL-6 that is resistant to suppression by CsA, was largely responsible for allograft fibrosis and limited transplant survival. Our data show that IL-3 induces allograft fibrosis and chronic rejection of heart transplants, and exerts its profibrotic effects by activation of infiltrating basophils. Blockade of IL-3 or basophil-derived cytokines may provide new strategies to prevent or delay the development of chronic allograft rejection.

Expression of IL-3 receptors and impact of IL-3 on human T and B cells.

A large number of animal models revealed that IL-3 plays an important role for the development of T and B cell-mediated autoimmune diseases. However, little is known about the expression and regulation of IL-3 receptors in human T and B cells and how IL-3 modulates the activation and survival of these cells. We show that the IL-3 receptor CD123 is substantially upregulated on proliferating CD4+ and CD8+ T as well as B cells. Upregulation of CD123 differs between various activators and can be further modulated by cytokines. Exposure of human T and B cells to IL-3 enhances proliferation and survival. IL-3 also induces a shift towards secretion of proinflammatory cytokines in T and B cells and reduces the expression of IL-10 in B cells. Thus IL-3 may have proinflammatory and immunostimulatory properties also in human autoimmune diseases.

Alloantigen specific deletion of primary human T cells by Fas ligand (CD95L)-transduced monocyte-derived killer-dendritic cells.

Numerous studies have been performed in vitro and in various animal models to modulate the interaction of dendritic cells (DC) and T cells by Fas (CD95/Apo-1) signalling to delete activated T cells via induction of activation-induced cell death (AICD). Previously, we could demonstrate that Fas ligand (FasL/CD95L)-expressing 'killer-antigen-presenting cells' can be generated from human monocyte-derived mature DC (mDC) using adenoviral gene transfer. To evaluate whether these FasL-expressing mDC (mDC-FasL) could eliminate alloreactive primary human T cells in vitro, co-culture experiments were performed. Proliferation of human T cells was markedly reduced in primary co-cultures with allogeneic mDC-FasL, whereas a strong proliferative T-cell response could be observed in co-cultures with enhanced green fluorescent protein-transduced mDC. Inhibition of T-cell proliferation was related to the transduction efficiency, and the numbers of mDC-FasL present in co-cultures. In addition, proliferation of pre-activated alloreactive CD4(+) and CD8(+) T cells could be almost completely inhibited in secondary co-cultures using mDC-FasL as stimulatory cells, which was the result of induction of apoptosis in the majority of preactivated T cells. The specific deletion of alloreactive T cells by mDC-FasL was confirmed by an unaffected proliferative response of surviving T cells towards allogeneic 'third-party' peripheral blood mononuclear cells in a third stimulation, or upon unspecific stimulation with anti-CD3/CD28 beads. The results of this study demonstrate that allospecifically activated T cells are efficiently eliminated by mDC-FasL, supporting further investigations to apply FasL-expressing 'killer-DC' as a novel strategy for the treatment of allograft rejection.

Peripheral Blood Mononuclear Cells: Isolation, Freezing, Thawing, and Culture.

The work with peripheral blood mononuclear cells (PBMCs), which comprise lymphocytes and monocytes, is indispensable in immunological diagnostics and research. The isolation of PBMCs takes advantage of differences in cell density of the different blood components. Density gradient centrifugation of diluted whole blood layered over a density gradient medium yields PBMCs; two subsequent washing steps remove remaining platelets. To store the cells for future assays, they can be frozen and thawed when required. Dimethyl sulfoxide (DMSO) serves as a cryoprotectant for freezing PBMCs, but must be removed by washing after thawing, as it can become toxic to the cells on longer exposure.

IL-3 promotes the development of experimental autoimmune encephalitis.

Little is known about the role of IL-3 in multiple sclerosis (MS) in humans and in experimental autoimmune encephalomyelitis (EAE). Using myelin oligodendrocyte glycoprotein (MOG) peptide-induced EAE, we show that CD4+ T cells are the main source of IL-3 and that cerebral IL-3 expression correlates with the influx of T cells into the brain. Blockade of IL-3 with monoclonal antibodies, analysis of IL-3 deficient mice, and adoptive transfer of leukocytes demonstrate that IL-3 plays an important role for development of clinical symptoms of EAE, for migration of leukocytes into the brain, and for cerebral expression of adhesion molecules and chemokines. In contrast, injection of recombinant IL-3 exacerbates EAE symptoms and cerebral inflammation. In patients with relapsing-remitting MS (RRMS), IL-3 expression by T cells is markedly upregulated during episodes of relapse. Our data indicate that IL-3 plays an important role in EAE and may represent a new target for treatment of MS.

Elimination of activated but not resting primary human CD4+ and CD8+ T cells by Fas ligand (FasL/CD95L)-expressing Killer-dendritic cells.

Dendritic cells (DC) genetically engineered to express high levels of Fas ligand (FasL/CD95L) have been demonstrated to delete T cells in an antigen specific manner in several different animal models in vivo. However, the immunomodulatory capacity of primary human FasL-expressing Killer-DC has not been determined. Therefore, human Killer-DC were generated from mature monocyte-derived DC using the inducible CRE/LoxP adenoviral vector system, and the immunoregulatory capacity of these cells was analyzed in cocultures with primary human T cells in vitro. Combined transductions of DC by AdloxPFasL and AxCANCre resulted in FasL expression in > 70% of DC without affecting the mature phenotype. Proliferation of activated primary human T cells was inhibited up to 80% in cocultures with FasL-expressing DC but not EGFP-transduced DC, which was due to induction of apoptosis in activated but not resting CD4+ and CD8+ T cells. Apoptosis induced by Killer-DC could be blocked by an anti-FasL-antibody in a dose dependent fashion. The present results demonstrate that FasL-expressing Killer-DC eliminate activated but not resting primary human CD4+ and CD8+ T cells by induction of Fas-mediated apoptosis supporting the concept to apply Killer-DC as a novel strategy for the treatment of T cell-dependent autoimmune disease and allograft rejection in humans.

A new flow cytometric assay for the simultaneous analysis of antigen-specific elimination of T cells in heterogeneous T cell populations.

Novel immunosuppressive strategies are targeting for an antigen-specific deletion of T cells responsible for organ damage in autoimmunity and allograft rejection. Here, we present a new flow cytometry-based assay that allows the reliable and efficient detection of T cells that were eliminated in an antigen-specific fashion. A stable cell-labelling technique utilizing the two membrane dyes PKH26 and PKH67 has been combined with annexin V and 7-aminoactinomycin (7-AAD) staining to detect apoptotic cells. A differential gating strategy enabled us to determine the viability/apoptosis for each PKH-stained T cell subpopulation independently. The capability to simultaneously analyze apoptosis within T cell mixtures of different antigen specificities establishes this assay as a superior tool for the further development of novel antigen-specific immunosuppressive approaches.

Killer artificial antigen-presenting cells: a novel strategy to delete specific T cells.

Several cell-based immunotherapy strategies have been developed to specifically modulate T cell-mediated immune responses. These methods frequently rely on the utilization of tolerogenic cell-based antigen-presenting cells (APCs). However, APCs are highly sensitive to cytotoxic T-cell responses, thus limiting their therapeutic capacity. Here, we describe a novel bead-based approach to modulate T-cell responses in an antigen-specific fashion. We have generated killer artificial APCs (kappaaAPCs) by coupling an apoptosis-inducing alpha-Fas (CD95) IgM mAb together with HLA-A2 Ig molecules onto beads. These kappaaAPCs deplete targeted antigen-specific T cells in a Fas/Fas ligand (FasL)-dependent fashion. T-cell depletion in cocultures is rapidly initiated (30 minutes), dependent on the amount of kappaaAPCs and independent of activation-induced cell death (AICD). kappaaAPCs represent a novel technology that can control T cell-mediated immune responses, and therefore has potential for use in treatment of autoimmune diseases and allograft rejection.

Monocyte-derived human macrophages mediate anergy in allogeneic T cells and induce regulatory T cells.

Activation of alloreactive T cells by APCs such as dendritic cells (DC) has been implicated as crucial step in transplant rejection. In contrast, it has been proposed that macrophages (Mphi) maintain tolerance toward alloantigens. It was therefore the aim of this study to further analyze the T cell-stimulatory capacity of mature DC and Mphi in vitro using the model of allogeneic MLR. There was a strong proliferative response in T cells cocultured with DC, which was further increased upon restimulation in a secondary MLR. In contrast, T cells did not proliferate in cocultures with Mphi despite costimulation with anti-CD28 and IL-2. Cytokine analysis revealed considerable levels of IL-10 in cocultures of T cells with Mphi, whereas high amounts of IL-2 and IFN-gamma were present in cocultures with DC. There was only minimal T cell proliferation in a secondary MLR when T cells were rescued from primary MLR with Mphi and restimulated with DC of the same donor, or DC of an unrelated donor (third party), whereas a strong primary proliferative response was observed in resting T cells, demonstrating induction of T cell anergy by Mphi. Functional analysis of T cells rescued from cocultures with Mphi demonstrated that anergy was at least partly mediated by IL-10-producing regulatory T cells induced by Mphi. These results demonstrate that Mphi drive the differentiation of regulatory T cells and mediate anergy in allogeneic T cells, supporting the concept that Mphi maintain peripheral tolerance in vivo.

Mature but not immature Fas ligand (CD95L)-transduced human monocyte-derived dendritic cells are protected from Fas-mediated apoptosis and can be used as killer APC.

Several in vitro and animal studies have been performed to modulate the interaction of APCs and T cells by Fas (CD95/Apo-1) signaling to delete activated T cells in an Ag-specific manner. However, due to the difficulties in vector generation and low transduction frequencies, similar studies with primary human APC are still lacking. To evaluate whether Fas ligand (FasL/CD95L) expressing killer APC could be generated from primary human APC, monocyte-derived dendritic cells (DC) were transduced using the inducible Cre/Loxp adenovirus vector system. Combined transduction of DC by AdLoxpFasL and AxCANCre, but not single transduction with these vectors, resulted in dose- and time-dependent expression of FasL in >70% of mature DC (mDC), whereas <20% of immature DC (iDC) expressed FasL. In addition, transduction by AdLoxpFasL and AxCANCre induced apoptosis in >80% of iDC, whereas FasL-expressing mDC were protected from FasL/Fas (CD95/Apo-1)-mediated apoptosis despite coexpression of Fas. FasL-expressing mDC eliminated Fas(+) Jurkat T cells as well as activated primary T cells by apoptosis, whereas nonactivated primary T cells were not deleted. Induction of apoptosis in Fas(+) target cells required expression of FasL in DC and cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. Induction of apoptosis in Fas(+) target cells required expression of FasL in DC, cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. The present results demonstrate that FasL-expressing killer APC can be generated from human monocyte-derived mDC using adenoviral gene transfer. Our results support the strategy to use killer APCs as immunomodulatory cells for the treatment of autoimmune disease and allograft rejection.